Methods and products for regulating lectin complement pathway associated complement activation

ABSTRACT

The invention relates to methods and products for regulating lectin complement pathway associated complement activation. The methods include both in vitro and in vivo methods for inhibiting lectin complement pathway associated complement activation. The methods are accomplished by contacting a mammalian cell having surface exposed MBL ligand with an effective amount of a mannan binding lectin inhibitor to inhibit lectin complement pathway associated complement activation. The mannan binding lectin inhibitor may be administered to a subject to prevent cellular injury mediated by lectin complement pathway associated complement activation. The products of the invention include compositions of a mannan binding lectin inhibitor. The mannan binding lectin inhibitor is an isolated mannan binding lectin binding peptide that selectively binds to a human mannan binding lectin epitope and that inhibits lectin complement pathway associated complement activation. The products also include hybridoma cell lines and pharmaceutical compositions.

RELATED APPLICATIONS

[0001] This application is a divisional application of U.S.Non-Provisional application Ser. No. 09/464,303, which claims priorityunder 35 U.S.C. §119 to U.S. Provisional Patent Application No.60/112,390, filed Dec. 15, 1998, the entire contents of which is herebyincorporated by reference.

GOVERNMENT SUPPORT

[0002] The present invention was supported in part by grants from theNational Institutes of Health HL56086, HL52886, and GM07592. The U.S.Government may retain certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to methods and products forregulating lectin complement pathway (LCP) associated complementactivation. In particular, the invention relates to methods forinhibiting LCP associated complement activation by contacting amammalian cell having a mannose binding lectin (MBL) ligand with an MBLinhibitor. The invention also relates to products which are MBLinhibitors, such as an MBL binding peptide.

BACKGROUND OF THE INVENTION

[0004] The immune system functions to defend the body against pathogenicbacteria, viruses and parasites. Immunity against foreign pathogensusually involves the complement system. The complement system is acascade of 18 sequentially activated serum proteins which functions torecruit and activate other cells of the immune system, effect cytolysisof target cells and induce opsonization of foreign pathogens. Complementcan be activated by the presence of either antibody/antigen complexes,as in the classical complement pathway, or microbial surfaces, as in thealternative complement pathway. Complement activation can also occur viathe lectin complement pathway (LCP). Lectins are carbohydrate-bindingproteins that recognize oligosaccharide structures present on cellsurfaces, the extracellular matrix, and secreted glycoproteins. As shownin FIG. 1, these distinct activation pathways ultimately converge at thecommon enzymatic step of serum protein C3 cleavage to C3b and C3a. Thisin turn initiates the terminal steps of complement function includingthe cleavage of C5 to C5b and C5a and subsequent deposition of C5b-C9onto the target cell membrane.

[0005] The LCP is an antibody-independent cascade that is initiated bybinding of mannan- (or mannose) binding lectin (MBL) to cell surfacecarbohydrates on bacteria, yeasts, parasitic protozoa, and viruses(Turner M W, “Mannose-binding lectin: The pluripotent molecule of theinnate immune system”, Immunol.Today, 1996;17:532-540). MBL (≈600 kDa)is a member of the collectin protein family and is structurally relatedto the classical complement C1 subcomponent, C1q. Associated with MBLare two serine proteases, Mannose binding lectin associated serineprotease, MASP-1 and MASP-2, which show striking homology to the twoC1q-associated serine proteases of the classical complement pathway, C1rand C1s (Thiel S, et al., “A second serine protease associated withmannan-binding lectin that activates complement”, Nature1997;386:506-510). The selectivity of MBL sugar binding is:N-acetyl-D-glucosamine (GluNAc)>mannose>N-acetylmannosamine andfucose>maltose>glucose>>galactose and N-acetylgalactosamine (Thiel S, etal., “A second serine protease associated with mannan-binding lectinthat activates complement”, Nature 1997;386:506-510; Turner M W,“Mannose-binding lectin: The pluripotent molecule of the innate immunesystem”, Immunol.Today, 1996;17:532-540). Binding of the MBL/MASPcomplex to cell surface carbohydrates activates the LCP, which in turnactivates the classical complement pathway independently of C1q, C1r,C1s or antibodies (FIG. 1). Most if not all the carbohydrate moieties towhich MBL binds are not normally expressed by unperturbed human tissue.

SUMMARY OF THE INVENTION

[0006] The present invention relates to methods and products forregulating lectin complement pathway (LCP) associated complementactivation. Prior to the instant invention, it was known that LCPassociated complement activation was a mechanism used by the body torecognize and destroy an invading microorganism. LCP activation normallyoccurs through the binding of mannan-binding lectin (MBL) and its twoassociated serine proteases, MASP-1 and MASP-2, to carbohydrates on thesurface of microorganisms. Once MBL and MASP-1 and MASP-2 are localizedto the surface of the microorganism, complement begins to assemble,ultimately killing the microorganism. These prior art teachingsdemonstrate that MBL is an important cellular component in the processof the eradication of infectious microorganisms. In fact, MBLdeficiencies can result in medical disorders. A disease known as MBLdeficiency, in which children are deficient in MBL, renders the childrenprone to the development of infectious diseases.

[0007] The present invention is based upon the surprising discovery thatMBL recognizes specific carbohydrates or peptides on the surface ofmammalian endothelial cells, causing complement deposition throughactivation of the LCP. According to U.S. Pat. No. 5,270,199 issued toEcekowitz, MBL does not recognize the cell wall of human and animalcells. In contrast to these prior art teachings, it has been discovered,according to the invention, that MBL does recognize specific sequenceson the surface of mammalian cells. It has also been discovered that MBLdeposition on the surface of mammalian cells results in activation ofLCP, contributing to the development of diseased or damaged tissue.

[0008] In one aspect, the invention is a method for inhibitingLCP-associated complement activation. The method includes the step ofcontacting a mammalian cell having a surface exposed MBL ligand with aneffective amount of an MBL inhibitor to inhibit cellular MBL depositionand LCP-associated complement activation. In one illustrativeembodiment, the method is an in vitro screening assay.

[0009] In another aspect, the invention is a method for inhibiting acellular injury mediated by LCP-associated complement activation. Themethod includes the step of administering to a subject in need thereofan effective amount of an MBL inhibitor to inhibit LCP-associatedcomplement activation.

[0010] In one embodiment of the methods of the invention, the MBLinhibitor is an isolated MBL binding peptide. In an illustrativeembodiment, the isolated MBL binding peptide has an MBL binding CDR3region or functional variant thereof. In some embodiments, the isolatedMBL binding peptide is an antibody fragment. In other embodiments, theisolated MBL binding peptide is an antibody.

[0011] According to another embodiment of the methods of the invention,the MBL inhibitor is an isolated MASP binding peptide. The isolated MASPbinding peptide may bind to either MASP-1 or MASP-2 or both, preventingMASP from participating in the LCP.

[0012] The cellular injury mediated by LCP-associated complementactivation may contribute to the development of injured tissueassociated with a variety of disorders. In one embodiment, the cellularinjury is associated with atherosclerosis. In another embodiment, thecellular injury is associated with arthritis, myocardial infarction,ischemia and reperfusion, transplantation, CPB, stroke, ARDS, SLE,Lupus, or dialysis.

[0013] The MBL inhibitor may be administered to the subject by any routeknown in the art. When the cellular injury is associated with thepulmonary system, the MBL inhibitor may be administered to the subjectby an aerosol route of delivery.

[0014] According to another aspect of the invention, an MBL inhibitor isprovided. The MBL inhibitor is an isolated peptide that selectivelybinds to a human MBL epitope and inhibits LCP-associated complementactivation.

[0015] In another aspect, the invention is a hybridoma cell line. In oneillustrative embodiment, the hybridoma cell line is the cell linedeposited under ATCC accession number HB-12621. In another embodiment,the hybridoma cell line is the cell line is deposited under ATCCaccession number HB-12620. In another embodiment, the hybridoma cellline is the cell line deposited under ATCC accession number HB-12619.

[0016] According to yet another aspect, the invention is a compositionof an MBL inhibitor, wherein the MBL inhibitor is an isolated bindingpeptide that selectively binds to a human MBL epitope and that inhibitsLCP-associated complement activation. In an illustrative embodiment thecomposition is a pharmaceutical composition including an effectiveamount for treating an MBL mediated disorder of the isolated MBL bindingpeptide and a pharmaceutically acceptable carrier. In one embodiment,the composition also includes a drug for the treatment of an MBLmediated disorder.

[0017] In one embodiment the isolated MBL binding peptide has an MBLbinding CDR3, region or a functional variant thereof of a monoclonalantibody produced by hybridoma cell line_(3F8) deposited under ATCCaccession number HB-12621. In another embodiment the isolated MBLbinding peptide has an MBL binding CDR3₂ region or a functional variantthereof of a monoclonal antibody produced by hybridoma cell line_(2A9)deposited under ATCC accession number HB-12620. In another embodimentthe isolated MBL binding peptide has an MBL binding CDR3₂ region or afunctional variant thereof of a monoclonal antibody produced byhybridoma cell line_(hMBL1.2) deposited under ATCC accession numberHB-12619.

[0018] The isolated peptide may be an intact soluble monoclonalantibody. In one embodiment the isolated peptide is monoclonalantibody_((3F8)) produced by the hybridoma cell line deposited underATCC Accession No. HB-12621. In another embodiment the isolated peptideis monoclonal antibody_((2A9)) produced by the hybridoma cell linedeposited under ATCC Accession No. HB-12620. In another embodiment theisolated peptide is monoclonal antibody_(hMBL1.2) produced by thehybridoma cell line deposited under ATCC Accession No. HB-12619. In anillustrative embodiment the isolated peptide is a humanized monoclonalantibody.

[0019] According to some embodiments the isolated peptide is an antibodyfragment. The isolated peptide, for instance, may be a monoclonalantibody fragment selected from the group consisting of an F(ab′)₂fragment, Fd fragment, and an Fab fragment. The isolated peptide mayalso be a peptide having a light chain CDR2 region selected from thegroup consisting of a CDR2_((3F8)) of a monoclonal antibody produced byhybridoma_((3F8)) deposited under ATCC Accession No. HB-12621, aCDR2_((2A9)) of a monoclonal antibody produced by hybridoma_(2A9)deposited under ATCC Accession No. HB-12620, and a CDR² _((hMBL1.2)) ofa monoclonal antibody produced by hybridoma_((hMBL1.2)) deposited underATCC Accession No. HB-12619. In another embodiment the isolated peptidehas a light chain CDR1 region selected from the group consisting of aCDR1_((3F8)) of a monoclonal antibody produced by hybridoma_((3F8))deposited under ATCC Accession No. HB-12621, a CDR1_((2A9)) of amonoclonal antibody produced by hybridoma_((2A9)) deposited under ATCCAccession No. HB-12620, and a CDR1_((hMBL1.2)) of a monoclonal antibodyproduced by hybridoma_((hMBLA.2)) deposited under ATCC Accession No.HB-12619.

[0020] In another aspect, the invention is a composition, wherein theMBL inhibitor is an anti-MBL antibody that: (i) selectively binds to ahuman MBL epitope, and (ii) prevents LCP activation.

[0021] In yet another aspect, the invention is a method for screening asubject for susceptibility to treatment with an MBL inhibitor. Themethod includes the steps of isolating a mammalian cell from a subject,and detecting the presence of an MBL on a surface of the mammalian cell,wherein the presence of the MBL indicates that the cell is susceptibleto LCP-associated complement activation and that the subject issusceptible to treatment with an MBL inhibitor. In one embodiment, themethod includes the step of contacting the MBL with a detection reagentthat selectively binds to the MBL to detect the presence of the MBL. Thedetection reagent in one embodiment is an isolated MBL binding peptide.

[0022] A method for screening a subject for susceptibility to treatmentwith MBL inhibitor is provided in another aspect of the invention. Themethod includes the steps of contacting a mammalian cell from a subjectwith a labeled isolated MBL binding peptide, and detecting the presenceof an MBL on the surface of the mammalian cell, wherein the presence ofthe MBL indicates that the cell is susceptible to LCP-associatedcomplement activation and that the subject is susceptible totreatment-with an MBL inhibitor. In one embodiment, the mammalian cellis an endothelial cell.

[0023] Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a schematic depicting the antigen/antibody-dependentclassical complement pathway and the antibody-independent alternativeand lectin complement pathways. All three pathways merge at C3 and leadto the formation of the terminal complement complex (C5b-9).

[0025]FIG. 2 depicts a flow cytometry printout to demonstrate MBLdeposition on HUVECs. MBL deposition on HUVECs subjected to zero(normoxia) or 24 hours of hypoxia was studied by flow cytometry using amonoclonal anti-human MBL antibody. MBL deposition (MFI=40±3) wassignificantly increased on hypoxic HUVECs reoxygenated for 3 hours in30% human serum compared to normoxic HUVECs (MFI=8±2), where MFI=meanfluorescent intensity.

[0026]FIG. 3 is a graph depicting MBL deposition on HUVECs (ELISA). MBLdeposition on HUVECs subjected to zero (normoxia) or 24 hours of hypoxiafollowed by 3 hours of reoxygenation was examined by ELISA using amonoclonal anti-human MBL antibody. MBL deposition on hypoxic HUVECsreoxygenated in the presence of 30% human serum (vehicle) wassignificantly greater than normoxic HUVECs or hypoxic HUVECsreoxygenated in 30% human serum treated with 30 mmol/L GluNac.

[0027]FIG. 4a is a graph depicting iC3b deposition following competitiveinhibition of MBL. iC3b deposition was studied by ELISA on HUVECsreoxygenated in the presence of 30% human serum treated with 30 mmol/LGluNAc, D-mannose, or L-mannose. Deposition of iC3b on hypoxic HUVECsreoxygenated in 30% human serum (vehicle) or 30% human serum treatedwith L-mannose was significantly greater than normoxic HUVECs. iC3bdeposition, however, on HUVECs reoxygenated in 30% human serum treatedwith GluNAc or D-mannose did not significantly differ from normoxiccontrols.

[0028]FIG. 4b is a graph depicting iC3b deposition following depletionof MBL from human serum. HUVECs were reoxygenated in the presence ofMBL-depleted human serum to inhibit the lectin complement pathway.Deposition of iC3b (ELISA) on hypoxic HUVECs reoxygenated in 30% humanserum was significantly greater (p<0.05) than normoxic HUVECs. iC3bdeposition, however, on hypoxic HUVECs reoxygenated in 30% MBL-depletedhuman cell was significantly less (p<0.05) than hypoxic HUVECsreoxygenated in 30% human serum. When MBL was added back to theMBL-depleted human serum, iC3b deposition on the hypoxic/reoxygenatedHUVECs was significantly greater than normoxic HUVECs.

[0029]FIG. 5 is a graph depicting percent hemolysis as an indicator ofclassical complement pathway activity. No significant differences in theserum complement hemolytic assay (CH₅₀) were observed between humanserum or MBL-depleted human serum, indicating that depletion of MBL didnot inhibit or deplete classical complement pathway activity;

[0030]FIG. 6 depicts a Western blot analysis of C3 activation followinghypoxia/reoxygenation using purified C2, C3, C4, and MBL. Western blotanalysis of the C3 and C3b α′-chain was performed under reducedconditions with a polyclonal anti-human C3 antibody on the supernatantsof normoxic and hypoxic (12 hours) HUVECs reoxygenated for 3 hours inthe presence of purified C2, C3, C4, and MBL. Lanes 1 and 2 representnormoxic HUVECs supernatant, lanes 3 and 4, hypoxic HUVECs supernatant,lane 5, purified C3 standard, and lane 6, purified C3b standard. Theresults demonstrate an increased band density of C3b α′-chain in thehypoxic/reoxygenated supernatants compared to the normoxic supernatants.The Figure is representative of five experiments.

[0031]FIG. 7 is a scan of a Western blot analysis of human MBL.Monoclonal antibodies 3F8, hMBL1.2, 2A9 or 1C10 were used for westernblot analysis of reduced MBL. Lanes 1, 2, 3 and 4 represent staining ofreduced human MBL with 10 μg/ml of mAb 2A9, hMBL1.2, 1C10 or 3F8,respectively. A single band with an approximate molecular weight (MW) of32 kDa (i.e., consistent with MBL) was observed with each mAb. Thisfigure is representative of three separate experiments.

[0032]FIG. 8 is a graph depicting C3 deposition with inhibitors.

[0033]FIG. 9 is a graph depicting inhibition of VCAM-1 expression.Reoxygenation of hypoxic HUVECs in 30% HS treated with PBS (Vehicle)induced a significant increase in VCAM-1 expression compared to normoxiccells incubated with 30% HS. Treatment of the 30% HS with 3F8 (5 μg/ml)significantly inhibited VCAM-1 expression. The bars represent the meanof 3 individual experiments. Brackets represent SEM. *represent p<0.05compared to the respective normoxia control. **represent p<0.05 comparedto vehicle treated hypoxia group.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The invention relates to methods and products for regulating andmanipulating lectin complement pathway (LCP)-associated complementactivation. As discussed above, the invention is based on the findingthat LCP-associated complement activation plays a role in complementinduced cellular injury of mammalian cells. It was discovered accordingto the invention that MBL interacts with carbohydrates or peptides onthe surface of mammalian cells in vitro and in vivo. The surfaceassociated MBL leads to the accumulation of complement on the surface ofthe cell, ultimately leading to cell injury or death. According to theprior art, LCP-associated complement activation was predominantlyassociated with infectious microorganisms, suggesting that MBLdeposition should be promoted in order to enhance the killing ofinfectious microorganisms. It was discovered, according to theinvention, that in mammals it is preferable to block MBL cellularassociation, preventing LCP-associated complement activation rather thanto promote it. The LCP is not necessary for eradication of infectiousmicroorganisms in adult mammals, and in fact, it contributes to cellularinjury associated with several types of disorders, such asatherosclerosis, arthritis, myocardial infarction, ischemia andreperfusion, transplantation, CPB, stroke, ARDS, SLE, Lupus, ordialysis.

[0035] In one aspect, the invention is a method for inhibitingLCP-associated complement activation. The method includes the steps ofcontacting a mammalian cell having surface exposed MBL ligand with aneffective amount of an MBL inhibitor to inhibit LCP-associatedcomplement activation.

[0036] The methods of the invention are useful for inhibitingLCP-associated complement activation on the surface of a mammalian cellhaving surface exposed MBL ligand (carbohydrate or peptide groups)recognized by MBL. The mammalian cell may be any cell in which the cellsurface carbohydrates or peptides interact with MBL. In one illustrativeembodiment, the mammalian cell is an endothelial cell having a surfaceexposed MBL ligand. For instance, vascular endothelial cells have beenshown in subjects that have sustained ischemic/reperfusion injury toexpress an MBL ligand. Mammalian cells having MBL ligands can easily beidentified. For instance, an MBL binding assay (e.g., such as thosedescribed below) can be used to identify MBL ligands.

[0037] The method for inhibiting LCP-associated complement activationmay be used for a variety of in vitro and in vivo purposes. The methodmay be used, for instance, as an in vitro screening assay. The in vitroscreening assay may be used to identify compounds which function as anMBL inhibitor, such as the assay described above, to identify mammaliancells having surface exposed MBL ligands, or to detect susceptibility ofa subject to treatment with MBL inhibitor. In order to screen a subjectfor susceptibility to treatment with an MBL inhibitor, a cell isisolated from the subject and the presence of MBL or the ability of MBLto bind to the surface is detected. If MBL is present on the surface ofa cell or is able to bind to the surface of a cell, then the cell issusceptible to LCP-associated complement activation. If this is thecase, then the subject is susceptible to treatment with an MBLinhibitor.

[0038] The methods of the invention are also useful in vivo when it isdesirable to inhibit MBL deposition on a mammalian cell surface. Forinstance, the methods of the invention are useful for treating an MBLmediated disorder. The MBL inhibitors can be used alone as a primarytherapy or in combination with other therapeutics as an adjuvant therapyto enhance the therapeutic benefits of other medical treatments.

[0039] The mammalian cell is contacted with an MBL inhibitor. The stepof “contacting” as used herein refers to the addition of the MBLinhibitor to a medium containing a mammalian cell. The medium may be anin vitro tissue culture or a biological specimen, an ex vivo sample, orin vivo. The step of contacting refers to the addition of the MBLinhibitor in such a manner that it will prevent LCP-associatedcomplement activation associated with the mammalian cell.

[0040] An “MBL mediated disorder” as used herein is a disorder whichinvolves cellular injury caused by LCP-associated complement activation.MBL disorders include, for instance, atherosclerosis, arthritis,myocardial infarction, ischemia and reperfusion, transplantation, CPB,stroke, ARDS, SLE, Lupus, or dialysis. Each of these disorders iswell-known in the art and is described, for instance, in Harrison'sPrinciples of Internal Medicine (McGraw Hill, Inc., New York).

[0041] Atherosclerosis and myocardial infarction can lead toischemia-reperfusion (I/R) injury. One of the underlying mechanisms forI/R-induced injury is the hypoxic and reoxygenated environments createdin affected tissues. Fluctuations in oxygen content as observed in theseinstances can create oxygen free radicals which have been reported to,among other things, modulate endothelial cell surface profile.

[0042] The invention also is useful for treating cellular injury arisingfrom ischemia/reperfusion associated with atherosclerosis and/orcardio-vascular remodeling. Injury to the vascular system can lead to anumber of undesirable health conditions, including, for example, formsof atherosclerosis and arteriosclerosis that are associated withunwanted vascular smooth muscle cell proliferation. A common injury tothe vascular system occurs as a side effect of a medical procedure fortreating ischemic heart disease. Ischemia refers to a lack of oxygen dueto inadequate perfusion of blood. Ischemic heart disease ischaracterized by a disturbance in cardiac function due to an inadequatesupply of oxygen to the heart. The most common form of this diseaseinvolves a reduction in the lumen of coronary arteries, which limitscoronary blood-flow. Under these conditions the carbohydrate or peptideresidues of the cell surface become exposed or an MBL ligand issynthesized, allowing MBL to associate with the cell surface andinitiate the LCP associated complement activation.

[0043] When ischemic heart disease becomes very serious, then managementmust be invasive. Until recently, ischemic heart disease was treated bycoronary-artery, bypass surgery. Less invasive procedures, however, nowhave been developed. These procedures involve the use of cathetersintroduced into the narrowed region of the blood vessel (“the stenosis”)for mechanically disrupting, laser ablating or dilating the stenosis.

[0044] The compositions may be administered in combination with othertherapeutic treatments. The most widely used method to achieverevascularization of a coronary artery is percutaneous transluminalcoronary angioplasty. A flexible guide wire is advanced into a coronaryartery and positioned across the stenosis. A balloon catheter then isadvanced over the guide wire until the balloon is positioned across thestenosis. The balloon then is repeatedly inflated until the stenosis issubstantially eliminated. This procedure, as compared to heart surgery,is relatively noninvasive and can result in hospital stays of only threedays. The procedure is an important tool in the management of seriousheart conditions.

[0045] An “MBL inhibitor” as used herein is a compound that preventsLCP-associated complement activation. The MBL inhibitor may function byblocking MBL deposition on the surface of a mammalian cell or byblocking the association of MASP-1 or MASP-2 or C3b associated with MBLdeposition. The ability of an MBL inhibitor to block MBL deposition orprevent association of MASP-1, MASP-2, or C3b with MBL can be detectedusing routine in vitro binding assays, such as the following assay (alsodescribed in the Examples).

[0046] MBL deposition (or association with MASP-1, MASP-2, or C3b) canbe measured by ELISA on normoxic HUVECs and HUVECs subjected to 24 hr ofhypoxia followed by 3 hr of reoxygenation in the presence of 30% humanserum (HS) or 30% HS treated with 3, 30, or 300 mmol/L ofN-acetyl-D-glucosamine (GluNAc) or with the putative binding peptide toinhibit competitively MBL deposition.

[0047] C3 and MBL specific cell surface ELISAs can be performed usingperoxidase-conjugated polyclonal goat anti-human C3 antibody (Cappel,West Chester, Pa.) and monoclonal anti-human MBL antibody (Biodesign,Kennebunk, Me., clone #131-1), respectively. HUVECs are grown toconfluence on 0.1% gelatinized 96-well plastic plates (Corning Costar,Cambridge, Mass.). The plates are then subjected to 0 (normoxia) or 24hr of hypoxia. Hypoxic stress is maintained using a humidified sealedchamber (Coy Laboratory Products, Inc., Grass Lake, Mich.) at 37° C.gassed with 1% O₂, 5% CO₂, balance N₂ (Collard C D, et al.,“Reoxygenation of hypoxic human umbilical vein endothelial cellsactivates the classical complement pathway”, Circulation,1997;96:326-333). Following the specified period of normoxia or hypoxia,the cell media are aspirated and 100 μl of one the following is added toeach well: 1) 30% HS, 2) Hank's balanced salt solution, 3) 30% HS+3, 30,or 300 mmol/L GluNAc, 4) 30% HS+3, 30, or 300 mmol/L D-mannose, 5) 30%HS +3, 30, 300 mmol/L L-mannose, 6) 30% MBL-depleted HS 7) 30%MBL-depleted HS+0.6 μg/ml MBL or 8) 30% HS+3, 30, or 300 mmol/L putativeMBL binding peptide. Additionally, 100 μl of purified MBL (3-300 ng/ml)is added to select wells to form a standard curve for quantitativeanalysis of MBL deposition. The cells are then reoxygenated for 3 hr at37° C. in 95% air and 5% CO₂. The cells are washed and then fixed with1% paraformaldehyde (Sigma Chemical Co., St. Louis, Mo.) for 30 min. Thecells are then washed and incubated at 4° C. for 1.5 hr with 50 μl ofperoxidase-conjugated polyclonal goat anti-human C3 antibody (1:1000dilution) or monoclonal anti-human MBL antibody (1:1000 dilution). TheMBL ELISA plates are then washed and incubated for 1 hr at 4° C. with 50μl of peroxidase-conjugated polyclonal goat anti-mouse IgG secondaryantibody (1:1000 dilution). After washing the cells, the plates aredeveloped with 50 μl of ABTS(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)), and read(Molecular Devices, Sunnyvale, Calif.) at 405 nm. Background controlsfor the C3 ELISA consist of cells to which only the anti-human C3antibody is added (i.e., no HS) or cells incubated with 30%heat-inactivated HS. Background controls for the MBL ELISA consist ofcells to which only secondary antibody and an isotype control monoclonalantibody to porcine C5a are added. Background optical density issubtracted from all groups. ELISA experiments are generally performed 3times using 6 wells per experimental group. Endothelial C3 and MBLdeposition on normoxic vs. hypoxic HUVECs is analyzed by two-wayanalysis of variance (ANOVA).

[0048] The MBL inhibitor prevents LCP-associated complement activation.Whether a particular compound can inhibit LCP-associated complementactivation can also be assessed using routine in vitro screening assays.For instance, the Complement hemolytic assay (CH₅₀) described in theExamples below can be performed on MBL-depleted HS in order todemonstrate that MBL depletion inhibit LCP-associated complementactivation. The assay may be performed, however, using MBL containing HSand adding an MBL binding peptide and/or a control peptide.

[0049] In one illustrative embodiment, the MBL inhibitor is an isolatedMBL binding peptide. An “isolated MBL binding peptide” as used herein isa peptide which binds to MBL and inhibits LCP associated complementactivation. One method by which MBL binding peptides inhibit LCPassociated complement activation is by binding to MBL and inhibiting MBLassociation with surface exposed MBL ligands. Additionally, the MBLbinding peptide may bind to MBL and inhibit the association between MBLand MASP-1 or -2 and/or C3b. Several peptides which bind to MBL or MASPhave been described in the art, including Lanzrein, A. S. et al.,“Mannan-binding lectin in human serum, cerebrospinal fluid and braintissue and its role in Alzheimer's disease”, Department of Pharmacology,University of Oxford, UK, May 11, 1998, Neuroreport, 9(7):1491-5; Jack,D. L. et al., “Activation of complement by mannose-binding lectin onisogenic mutants of Neisseria meningitidis serogroup B”, ImmunobiologyUnit, Institute of Child Health, London, UK, J Immunol, Feb. 1,1998,160(3):1346-53, Terai, l. et al., “Human serum mannose-bindinglectin (MBL)-associated serine protease-1 (MASP-1): determination oflevels in body fluids and identification of two forms in serum”,Division of Clinical Pathology, Hokkaido Institute of Public Health,Sapporo, Japan, Clin. Exp. Immunol., November, 1997, 110(2):317-23;Endo, M. et al., “Glomerular deposition of mannose-binding lectin (MBL)indicates a novel mechanism of complement activation in IgA nephropathy[In Process Citation]”, Second Department of Internal Medicine, NihonUniversity School of Medicine, Tokyo, Japan, Nephrol Dial Transplant,Aug. 13, 1998, (8):1984-90; Valdimarsson, H. et al., “Reconstitution ofopsonizing activity by infusion of mannan-binding lectin (MBL) toMBL-deficient humans”, Department of Immunology, University ofReykjavik, Iceland, Scand. J Immunol., August 1998, 48(2):116-23; Thiel,S. et al., “The concentration of the C-type lectin, mannan-bindingprotein, in human plasma increases during an acute phase response”, ClinExp. Immunol., October 1992, 90(1):31-5. These peptides can be testedfor their ability to inhibit the association between MBL and MASP-1 or-2 and/or C3b.

[0050] The preferred compositions of the invention include an MBLinhibitor which is an isolated binding peptide that selectively binds toa human MBL epitope and that inhibits LCP-associated complementactivation. A “human MBL epitope” as used herein is a portion of MBLwhich when contacted with an MBL-binding peptide inhibits LCP-associatedcomplement activation by preventing the association between MBL and theMBL ligand or MASP-1 or -2 and/or C3b. Preferably the MBL epitope is aregion of the MBL which interacts with any of the three depositedmonoclonal antibodies.

[0051] In another embodiment, the MBL inhibitor is an isolated MASPbinding peptide. An “isolated MASP binding peptide” as used hereinrefers to a peptide which binds to MASP-1 or MASP-2 and preventsLCP-associated complement activation by preventing MASP-1 or MASP-2 fromforming a complex with MBL on the surface of a cell thereby preventingthe resulting C3b deposition associated with the MBL-MASP complex.

[0052] In another embodiment the MBL inhibitor is a mannan bindingpeptide. A “mannan binding peptide” as used herein is a peptide whichbinds to the MBL ligand on the surface of a mammalian cell, preventingits interaction with the MBL-MASP complex. The MBL inhibitors may easilybe prepared or identified by those of ordinary skill in the art usingroutine experiments since MBL, MASP, mannan and C3b are all well knowncompounds which have been characterized and described extensively in theprior art.

[0053] The MBL, MASP, and mannan binding peptides of the invention canbe identified using routine assays, such as the binding and LCPcomplement activation assays described above and elsewhere throughoutthis patent application.

[0054] The peptides of the invention are isolated peptides. As usedherein, with respect to peptides, the term “isolated peptides” meansthat the peptides are substantially pure and are essentially free ofother substances with which they may be found in nature or in vivosystems to an extent practical and appropriate for their intended use.In particular, the peptides are sufficiently pure and are sufficientlyfree from other biological constituents of their hosts cells so as to beuseful in, for example, producing pharmaceutical preparations orsequencing. Because, an isolated peptide of the invention may be admixedwith a pharmaceutically acceptable carrier in a pharmaceuticalpreparation, the peptide may comprise only a small percentage by weightof the preparation. The peptide is nonetheless substantially pure inthat it has been substantially separated from the substances with whichit may be associated in living systems.

[0055] MBL binding peptides also may easily be synthesized or producedby recombinant means by those of skill in the art. Methods for preparingor identifying peptides which bind to a particular target are well knownin the art. Molecular imprinting, for instance, may be used for the denovo construction of macromolecular structures such as peptides whichbind to a particular molecule. See for example Kenneth J. Shea,Molecular Imprinting of Synthetic Network Polymers: The De Novosynthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2,No. 5, May 1994; Klaus Mosbach, Molecular Imprinting, Trends in Biochem.Sci., 19(9) January 1994; and Wulff, G., in Polymeric Reagents andCatalysts (Ford, W. T., Ed.) ACS Symposium Series No. 308, pp 186-230,American Chemical Society (1986). One method for preparing mimics of MBLbinding peptides involves the steps of: (i) polymerization of functionalmonomers around a known MBL binding peptide or the binding region of ananti-MBL antibody (such as the deposited antibodies) (the template) thatexhibits a desired activity; (ii) removal of the template molecule; andthen (iii) polymerization of a second class of monomers in the void leftby the template, to provide a new molecule which exhibits one or moredesired properties which are similar to that of the template. Inaddition to preparing peptides in this manner other MBL bindingmolecules which are MBL inhibitors such as polysaccharides, nucleosides,drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins,steroids, lipids, and other biologically active materials can also beprepared. This method is useful for designing a wide variety ofbiological mimics that are more stable than their natural counterparts,because they are typically prepared by the free radical polymerizationof functional monomers, resulting in a compound with a nonbiodegradablebackbone. Other methods for designing such molecules include for exampledrug design based on structure activity relationships which require thesynthesis and evaluation of a number of compounds and molecularmodeling.

[0056] Peptides which bind to the MBL may also be identified byconventional screening methods such as phage display procedures (e.g.,methods described in Hart, et al., J. Biol. Chem. 269:12468 (1994)).Hart et al. report a filamentous phage display library for identifyingnovel peptide ligands for mammalian cell receptors. In general, phagedisplay libraries using, e.g., M13 or fd phage, are prepared usingconventional procedures such as those described in the foregoingreference. The libraries display inserts containing from 4 to 80 aminoacid residues. The inserts optionally represent a completely degenerateor a biased array of peptides. Ligands that bind selectively to MBL areobtained by selecting those phages which express on their surface aligand that binds to the MBL. These phages then are subjected to severalcycles of reselection to identify the peptide ligand-expressing phagesthat have the most useful binding characteristics. Typically, phagesthat exhibit the best binding characteristics (e.g., highest affinity)are further characterized by nucleic acid analysis to identify theparticular amino acid sequences of the peptides expressed on the phagesurface and the optimum length of the expressed peptide to achieveoptimum binding to the MBL. Alternatively, such peptide ligands can beselected from combinatorial libraries of peptides containing one or moreamino acids. Such libraries can further be synthesized which containnon-peptide synthetic moieties which are less subject to enzymaticdegradation compared to their naturally-occurring counterparts.

[0057] To determine whether a peptide binds to MBL any known bindingassay may be employed. For example, the peptide may be immobilized on asurface and then contacted with a labeled MBL. The amount of MBL whichinteracts with the peptide or the amount which does not bind to thepeptide may then be quantitated to determine whether the peptide bindsto MBL. A surface having the deposited monoclonal antibody immobilizedthereto may serve as a positive control.

[0058] Screening of peptides of the invention, also can be carried oututilizing a competition assay. If the peptide being tested competes withthe deposited monoclonal antibody, as shown by a decrease in binding ofthe deposited monoclonal antibody, then it is likely that the peptideand the deposited monoclonal antibody bind to the same, or a closelyrelated, epitope. Still another way to determine whether a peptide hasthe specificity of the deposited monoclonal antibody of the invention isto pre-incubate the deposited monoclonal antibody with MBL with which itis normally reactive, and then add the peptide being tested to determineif the peptide being tested is inhibited in its ability to bind MBL. Ifthe peptide being tested is inhibited then, in all likelihood, it hasthe same, or a functionally equivalent, epitope and specificity as thedeposited monoclonal antibody.

[0059] Using routine procedures known to those of ordinary skill in theart, one can determine whether a peptide which binds to MBL is usefulaccording to the invention by determining whether the peptide is onewhich blocks MBL from binding to an MBL ligand. Such assays aredescribed above and in the Examples section. Other assays will beapparent to those of skill in the art, having read the presentspecification, which are useful for determining whether a peptide whichbinds to MBL also inhibitors LCP associated complement activation.

[0060] By using the deposited monoclonal antibodies of the invention, itis now possible to produce anti-idiotypic antibodies which can be usedto screen other antibodies to identify whether the antibody has the samebinding specificity as the deposited monoclonal antibodies of theinvention. In addition, such anti-idiotypic antibodies can be used foractive immunization (Herlyn, et al., Science, 232:100, 1986). Suchanti-idiotypic antibodies can be produced using well-known hybridomatechniques (Kohler and Milstein, Nature, 256:495, 1975). Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the deposited monoclonal antibodies. Thesedeterminants are located in the hypervariable region of the antibody. Itis this region which binds to a given epitope and, thus, is responsiblefor the specificity of the antibody. An anti-idiotypic antibody can beprepared by immunizing an animal with the deposited monoclonalantibodies. The immunized animal will recognize and respond to theidiotypic determinants of the immunizing deposited monoclonal antibodiesand produce an antibody to these idiotypic determinants. By using theanti-idiotypic antibodies of the immunized animal, which are specificfor the deposited monoclonal antibodies of the invention, it is possibleto identify other clones with the same idiotype as the depositedmonoclonal antibody used for immunization. Idiotypic identity betweenmonoclonal antibodies of two cell lines demonstrates that the twomonoclonal antibodies are the same with respect to their recognition ofthe same epitopic determinant. Thus, by using anti-idiotypic antibodies,it is possible to identify other hybridomas expressing monoclonalantibodies having the same epitopic specificity.

[0061] It is also possible to use the anti-idiotype technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is theimage of the epitope bound by the first monoclonal antibody. Thus, theanti-idiotypic monoclonal antibody can be used for immunization, sincethe anti-idiotype monoclonal antibody binding domain effectively acts asan antigen.

[0062] Activation assays also can be used to assess the relativeinhibitory concentrations of a peptide in an activation assay and toidentify those peptides which inhibit activation by at least, e.g., 75%.

[0063] Other assays will be apparent to those of skill in the art,having read the present specification, which are useful for determiningwhether a peptide which binds to MBL also inhibits MBL activation.

[0064] In one embodiment the peptide that inhibits the activation of MBLis an antibody or a functionally active antibody fragment. Antibodiesare well known to those of ordinary skill in the science of immunology.As used herein, the term “antibody” means not only intact antibodymolecules but also fragments of antibody molecules retaining MBL bindingability. Such fragments are also well known in the art and are regularlyemployed both in vitro and in vivo. In particular, as used herein, theterm “antibody” means not only intact immunoglobulin molecules but alsothe well-known active fragments F(ab′)₂, and Fab. F(ab′)₂, and Fabfragments which lack the Fc fragment of intact antibody, clear morerapidly from the circulation, and may have less non-specific tissuebinding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)). As is well-known in the art, the complementarity determiningregions (CDRs) of an antibody are the portions of the antibody which arelargely responsible for antibody specificity. The CDR's directlyinteract with the epitope of the antigen (see, in general, Clark, 1986;Roitt, 1991). In both the heavy chain and the light chain variableregions of IgG immunoglobulins, there are four framework regions (FR1through FR4) separated respectively by three complementarity determiningregions (CDR1 through CDR3). The framework regions (FRs) maintain thetertiary structure of the paratope, which is the portion of the antibodywhich is involved in the interaction with the antigen. The CDRs, and inparticular the CDR3 regions, and more particularly the heavy chain CDR3contribute to antibody specificity. Because these CDR regions and inparticular the CDR3 region confer antigen specificity on the antibodythese regions may be incorporated into other antibodies or peptides toconfer the identical antigen specificity onto that antibody or peptide.

[0065] As discussed above the MBL inhibitors of the present inventionencompass in some embodiments of the invention MBL binding peptideswhich include a MBL binding region which specifically binds to human MBLand inhibits LCP associated complement activation, e.g., by preventingMBL from interacting with MBL ligands. “MBL ligands” as used herein arecarbohydrates or peptides with which MBL can interact. Optionally theMBL binding region is a MBL binding CDR3 region. A “MBL binding CDR3region” as used herein is a CDR3 peptide sequence derived from themonoclonal antibodies produced by the hybridomas deposited with the ATCCunder ATCC Accession No. (HB-12621), ATCC Accession No. (HB-12620), andATCC Accession No. (HB-12619).

[0066] Three antibody producing hybridoma cell lines (3F8, 2A9, hMBL1.2)were deposited by Applicants with the ATCC on Dec. 15, 1998. Hybridoma3F8 produces monoclonal antibody_((3F8)) having binding specificity forMBL. Monoclonal antibody_(3F8) includes the CDR3_(3F8) region within itssequence. As used herein “CDR3_((3F8))” includes the CDR3 region ofmonoclonal antibody_((3F8)). Hybridoma_((2A9)) produces monoclonalantibody_((2A9)) having binding specificity for MBL. Monoclonalantibody_((2A9)) includes the CDR3_((2A9)) region within its sequence.As used herein “CDR3_((2A9))” includes the CDR3 region of monoclonalantibody_(2A9). Hybridoma_((hMBL1.2)) produces monoclonalantibody_((hMBL1.2)) having binding specificity for MBL. Monoclonalantibody_((hMBL1.2)) includes the CDR³ _((hMBL1.2)) region within itssequence. As used herein “CDR³ _((hMBL1.2))” includes the CDR3 region ofmonoclonal antibody_((hMBL1.2)). Each of monoclonal antibody_(3F8),monoclonal antibody_(2A9), and monoclonal antibody_((hMBL1.2))specifically bind to MBL and prevent MBL from interacting with an MBLligand.

[0067] The “MBL binding CDR3 region” refers to the CDR3_((3F8)),CDR3_((2A9)) and CDR3_((hMBL1.2)) peptide sequences. In one embodimentthe peptides of the invention include functional variants of the MBLbinding CDR3 region. A “functional variant” as used herein is a peptidehaving the sequence of the CDR3_((3F8)), CDR3_((2A9)), orCDR3_((hMBL1.2)) regions with conservative substitutions therein. Asused herein, “conservative substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the peptide in which the amino acid substitution ismade. Conservative substitutions of amino acids include substitutionsmade amongst amino acids with the following groups: (1) M,I,L,V; (2)F,Y,W; (3) K,R,H; (4) A,G; (5) S,T; (6) Q,N; and, (7) E,D. Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino-acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding the CDR3 region.These and other methods for altering a CDR3 region peptide will be knownto those of ordinary skill in the art and may be found in referenceswhich compile such methods, e.g. Sambrook. et al., Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press,1989. The activity of functionally equivalent variants of the MBLbinding CDR3 region can be tested by the binding and activity assaysdiscussed above.

[0068] For purposes of brevity the term “ATCC deposited hybridoma” isused throughout the specification to refer to the three hybridomasdeposited with the ATCC on Dec. 15, 1998. The term “deposited monoclonalantibody” is used to refer to each of the monoclonal antibodies(monoclonal antibody_((3F8)), monoclonal antibody_((2A9)), or monoclonalantibody_((hMBL1.2)) produced by the ATCC deposited hybridomas. Forpurposes of definiteness in the attached claims each of the hybridomasand monoclonal antibodies is specifically recited.

[0069] According to one embodiment, the peptide of the invention is anintact soluble anti-MBL monoclonal antibody in an isolated form or in apharmaceutical preparation. An intact soluble monoclonal antibody, as iswell known in the art, is an assembly of polypeptide chains linked bydisulfide bridges. Two principle polypeptide chains, referred to as thelight chain and heavy chain, make up all major structural classes(isotypes) of antibody. Both heavy chains and light chains are furtherdivided into subregions referred to as variable regions and constantregions. As used herein the term “monoclonal antibody” refers to ahomogenous population of immunoglobulins which specifically bind to anepitope (i.e. antigenic determinant) of human MBL.

[0070] The peptide of the invention in one embodiment is, for example,the deposited monoclonal antibody. The preparation and use of thedeposited monoclonal antibody is described more fully in the attachedExamples. In another embodiment the peptide of the invention is anintact antibody having the binding characteristics of the depositedmonoclonal antibody. An antibody having the binding characteristics ofthe deposited monoclonal antibody is one which binds to MBL and inhibitsMBL from interacting with MBL ligands. One of ordinary skill in the artcan easily identify antibodies having the binding characteristics of thedeposited monoclonal antibody using the screening and binding assays setforth in detail below.

[0071] In one set of embodiments, the peptide useful according to themethods of the present invention is an intact humanized anti-MBLmonoclonal antibody in an isolated form or in a pharmaceuticalpreparation. The following examples of methods for preparing humanizedmonoclonal antibodies that interact with MBL and inhibit LCP associatedcomplement activation are exemplary and are provided for illustrativepurposes only.

[0072] A “humanized monoclonal antibody” as used herein is a humanmonoclonal antibody or functionally active fragment thereof having humanconstant regions and a MBL binding CDR3 region from a mammal of aspecies other than a human. Humanized monoclonal antibodies may be madeby any method known in the art. Humanized monoclonal antibodies, forexample, may be constructed by replacing the non-CDR regions of anon-human mammalian antibody with similar regions of human antibodieswhile retaining the epitopic specificity of the original antibody. Forexample, non-human CDRs and optionally some of the framework regions maybe covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. There are entities in the United States which willsynthesize humanized antibodies from specific murine antibody regionscommercially, such as Protein Design Labs (Mountain View Calif.).

[0073] European Patent Application 0239400, the entire contents of whichis hereby incorporated by reference, provides an exemplary teaching ofthe production and use of humanized monoclonal antibodies in which atleast the CDR portion of a murine (or other non-human mammal) antibodyis included in the humanized antibody. Briefly, the following methodsare useful for constructing a humanized CDR monoclonal antibodyincluding at least a portion of a mouse CDR. A first replicableexpression vector including a suitable promoter operably linked to a DNAsequence encoding at least a variable domain of an Ig heavy or lightchain and the variable domain comprising framework regions from a humanantibody and a CDR region of a murine antibody is prepared. Optionally asecond replicable expression vector is prepared which includes asuitable promoter operably linked to a DNA sequence encoding at leastthe variable domain of a complementary human Ig light or heavy chainrespectively. A cell line is then transformed with the vectors.Preferably the cell line is an immortalized mammalian cell line oflymphoid origin, such as a myeloma, hybridoma, trioma, or quadroma cellline, or is a normal lymphoid cell which has been immortalized bytransformation with a virus. The transformed cell line is then culturedunder conditions known to those of skill in the art to produce thehumanized antibody.

[0074] As set forth in European Patent Application 0239400 severaltechniques are well known in the art for creating the particularantibody domains to be inserted into the replicable vector. (Preferredvectors and recombinant techniques are discussed in greater detailbelow.) For example, the DNA sequence encoding the domain may beprepared by oligonucleotide synthesis. Alternatively a synthetic genelacking the CDR regions in which four framework regions are fusedtogether with suitable restriction sites at the junctions, such thatdouble stranded synthetic or restricted subcloned CDR cassettes withsticky ends could be ligated at the junctions of the framework regions.Another method involves the preparation of the DNA sequence encoding thevariable CDR containing domain by oligonucleotide site-directedmutagenesis. Each of these methods is well known in the art. Therefore,those skilled in the art may construct humanized antibodies containing amurine CDR region without destroying the specificity of the antibody forits epitope.

[0075] In preferred embodiments, the humanized antibodies of theinvention are human monoclonal antibodies including at least the MBLbinding CDR3 region of the deposited monoclonal antibody. As notedabove, such humanized antibodies may be produced in which some or all ofthe FR regions of deposited monoclonal antibodies have been replaced byhomologous human FR regions. In addition, the Fc portions may bereplaced so as to produce IgA or IgM as well as human IgG antibodiesbearing some or all of the CDRs of the deposited monoclonal antibody. Ofparticular importance is the inclusion of the deposited monoclonalantibody MBL binding CDR3 region and, to a lesser extent, the other CDRsand portions of the framework regions of the deposited monoclonalantibody. Such humanized antibodies will have particular clinicalutility in that they will specifically recognize human MBL but will notevoke an immune response in humans against the antibody itself. In amost preferred embodiment, a murine CDR is grafted into the frameworkregion of a human antibody to prepare the “humanized antibody.” See,e.g., L. Riechmann et al., Nature 332, 323 (1988); M. S. Neuberger etal., Nature 314, 268 (1985) and EPA 0 239 400 (published Sep. 30, 1987).

[0076] In addition to the deposited monoclonal antibodies, otherantibodies (e.g., anti-MBL, anti-MASP, anti-mannan-like antibodies) canbe generated. The following is a description of a method for developinga monoclonal antibody specific for MBL (MASP-1 or -2, or mannan). Thedescription is exemplary and is provided for illustrative purposes only.

[0077] Murine monoclonal antibodies may be made by any of the methodsknown in the art utilizing MBL as an immunogen. An example of a methodfor producing murine monoclonals useful according to the invention isthe following: Female Balb/C mice were initially inoculated (i.p.) with250 ul of the following mixture: 250 μl Titermax mixed with 100 μg humanMBL in 250 μl PBS. The following week and for three consecutive weeksthe mice were injected with 50 μg hMBL in 250 μl PBS. On the 4th weekthe mice were injected with 25 μg MBL in 250 μl PBS and the mice werefused 4 days later.

[0078] The fusion protocol is adapted from Current Protocols inImmunology. The splenocytes were fused 1:1 with myelinoma fusion partnerP301 from ATCC using PEG 150 at 50% w/v. The fused cells were plated ata density of 1.25×10⁶/m. with 100 μl/well of a 96 well microtiter plate.The fusion media consisted of Deficient DME high glucose, Pen/Strep(50,000 U pen, 50,000 μg strep per liter), 4 mM L-glutamine, 20% fetalbovine serum, 10% thyroid enriched media, 1% OPI, 1% NEAA, 1% HAT, and50 μM 2-mercaptoethanol. The cells were fed 100 μl/well on day one and100/well media were exchanged on days 2, 3, 4, 7, 9, 11, and 13. Thelast media change before primary screening consisted of HAT substitutedfor the 1% HT. All subsequent feedings were done with fusion media minusthe minus HT or HAT. Screening was done with human MBL plated to plasticELISA plates (96 well plates). Purified hMBL was plated in each well at50 μl volume containing 2 μg/ml MBL in 2% sodium carbonate buffer. Theplates were then blocked with 3% BSA in PBS. Tissue culture media (50μl) was placed in the wells and incubated for 1 hour at roomtemperature. The plates were washed and a secondary HRP labeled goatanti-mouse IgG antibody was used for detection. Colorimetric analysiswas done with ABTS and read at a405 nm. Positive controls consisted of apolyclonal antibody to human MBL. Cells are then grown in mediaconsisting of the following: DMEM high glucose no-I-glut, sod, pyruvate500 ml (Irvine Scientific #9024), heat inactivated Hyclone 10%, 1%Non-essential amino acids, 4 mM L-gluamine, 100 U/ml penicillin and 100μg/ml streptomycin. All positive wells were then screened for functionin a secondary screen.

[0079] Human monoclonal antibodies may be made by any of the methodsknown in the art, such as those disclosed in U.S. Pat. No. 5,567,610,issued to Borrebaeck et al., U.S. Pat. No. 565,354, issued to Ostberg,U.S. Pat. No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133:3001 (1984), Brodeur, et al., Monoclonal Antibody Production Techniquesand Applications, p. 51-63 (Marcel Dekker, Inc, new York, 1987), andBoerner el al., J. Immunol., 147: 86-95 (1991). In addition to theconventional methods for preparing human monoclonal antibodies, suchantibodies may also be prepared by immunizing transgenic animals thatare capable of producing human antibodies (e.g., Jakobovits et al., PNASUSA, 90: 2551 (1993), Jakobovits et al., Nature, 362: 255-258 (1993),Bruggermann et al., Year in Immuno., 7:33 (1993) and U.S. Pat. No.5,569,825 issued to Lonberg).

[0080] An example of one method for producing human monoclonals usefulaccording to the invention is the following: Peripheral BloodLymphocytes (PBL) are isolated from healthy human donors using densitycentrifugation, and further separated into B, T and accessory (A) cells,described methods such as (Danielsson, L., Moller, S. A. & Borrebaeck,C. A. K. Immunology 61, 51-55 (1987)). PBL are fractionated into T andnon-T cells by rosetting with 2-amino ethyl (isothiouroniumbromide)—treated sheep red corpuscles, and the latter cell population isincubated on Petri dishes coated with fibronectin or autologous plasma.Non-adherent cells (B-cells) are decanted, and adherent cells (accessorycells) are removed by 10 mM EDTA. The B cells are stimulated with 50 μgStaphylococcus aureus Cowan l/ml and irradiated (2000R) T cells with 10μg PWM/ml overnight. The accessory cells are stimulated with 5 IU gammainterferon/ml and 10 μm indomethacin. The cell populations are culturedin supplemented RPMI 1640 which contains 10% human AB serum at a cellratio of 2:1:0.4 (Ti:B:A) for a total of 6 days. The antigenic dose ofMBL is 1 μg/ml. The culture is supplemented with recombinant IL-2 (5U/ml) and sPWM-T (25% by vol.), produced by described methods such as(Danielsson, L., Moller, S. A. & Borrebaeck C. A. K. Immunology 61,51-55 (1987)). T cells (10 cells/ml) suspended in serum-free RPMI 1640are incubated with 2.5 mM freshly prepared Leu-OMe for 40 min at roomtemperature. The cells are then washed 3 times in RPMI 1640 containing2% human antibody serum.

[0081] In one embodiment of the invention the peptide containing a MBLbinding region is a functionally active antibody fragment.Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions of theantibody, for example, are effectors of the complement cascade but arenot involved in antigen binding. An antibody from which the pFc′ regionhas been enzymatically cleaved, or which has been produced without thepFc′ region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. An isolated F(ab′)₂ fragment isreferred to as a bivalent monoclonal fragment because of its two antigenbinding sites. Similarly, an antibody from which the Fc region has beenenzymatically cleaved, or which has been produced without the Fc region,designated an Fab fragment, retains one of the antigen binding sites ofan intact antibody molecule. Proceeding further, Fab fragments consistof a covalently bound antibody light chain and a portion of the antibodyheavy chain denoted Fd (heavy chain variable region). The Fd fragmentsare the major determinant of antibody specificity (a single Fd fragmentmay be associated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation. The terms Fab, Fc, pFc′, F(ab′)2 and Fv are used consistentlywith their standard immunological meanings [Klein, Immunology (JohnWiley, New York, N.Y., 1982); Clark, W. R. (1986) The ExperimentalFoundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt,I. (1991) Essential Immunology, 7th Ed., (Blackwell ScientificPublications, Oxford)].

[0082] As used herein the term “functionally active antibody fragment”means a fragment of an antibody molecule including a MBL binding peptideof the invention which retains the LCP associated complement inhibitoryactivity of an intact antibody having the same specificity such as thedeposited monoclonal antibodies. Such fragments are also well known inthe art and are regularly employed both in vitro and in vivo. Inparticular, well-known functionally active antibody fragments includebut are not limited to F(ab′)₂, Fab, Fv and Fd fragments of antibodies.These fragments which lack the Fc fragment of intact antibody, clearmore rapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325(1983)). For example, single-chain antibodies can be constructed inaccordance with the methods described in U.S. Pat. No. 4,946,778 toLadner et al. Such single-chain antibodies include the variable regionsof the light and heavy chains joined by a flexible linker-moiety.Methods for obtaining a single domain antibody (“Fd”) which comprises anisolated variable heavy chain single domain, also have been reported(see, for example, Ward et al., Nature 341:644-646 (1989), disclosing amethod of screening to identify an antibody heavy chain variable region(V_(H) single domain antibody) with sufficient affinity for its targetepitope to bind thereto in isolated form). Methods for makingrecombinant Fv fragments based on known antibody heavy chain and lightchain variable region sequences are known in the art and have beendescribed, e.g., Moore et al., U.S. Pat. No. 4,462,334. Other referencesdescribing the use and generation of antibody fragments include e.g.,Fab fragments (Tijssen, Practice and Theory of Enzyme Immunoassays(Elsevieer, Amsterdam, 1985)), Fv fragments (Hochman et al.,Biochemistry 12: 1130 (1973); Sharon et al., Biochemistry 15: 1591(1976); Ehrilch et al., U.S. Pat. No. 4,355,023) and portions ofantibody molecules (Audilore-Hargreaves, U.S. Pat. No. 4,470,925). Thoseskilled in the art may construct antibody fragments from variousportions of intact antibodies without destroying the specificity of theantibodies for the MBL epitope.

[0083] Functionally active antibody fragments also encompass “humanizedantibody fragments.” As one skilled in the art will recognize, suchfragments can be prepared by traditional enzymatic cleavage of intacthumanized antibodies. If, however, intact antibodies are not susceptibleto such cleavage, because of the nature of the construction involved,the noted constructions can be prepared with immunoglobulin fragmentsused as the starting materials; or, if recombinant techniques are used,the DNA sequences, themselves, can be tailored to encode the desired“fragment” which, when expressed, can be combined in vivo or in vitro,by chemical or biological means, to prepare the final desired intactimmunoglobulin fragment.

[0084] In addition to the identification of peptides from libraries etc.the peptides of the invention including those containing the MBL bindingCDR3 region may easily be synthesized or produced by recombinant means.Such methods are well known to those of ordinary skill in the art.Peptides can be synthesized for example, using automated peptidesynthesizers which are commercially available. The peptides can beproduced by recombinant techniques by incorporating the DNA expressingthe peptide into an expression vector and transforming cells with theexpression vector to produce the peptide.

[0085] The sequence of the CDR regions, for instance, for use insynthesizing peptides of the invention, may be determined by methodsknown in the art. The heavy chain variable region is a peptide whichgenerally ranges from 100 to 150 amino acids in length. The light chainvariable region is a peptide which generally ranges from 80 to 130 aminoacids in length. The CDR sequences within the heavy and light chainvariable regions which include only approximately 3-25 amino acidsequences may easily be sequenced by one of ordinary skill in the art.The peptides may even be synthesized by commercial sources such as bythe Scripps Protein and Nucleic Acids Core Sequencing Facility (La JollaCalif.).

[0086] The sequences responsible for the specificity of the depositedmonoclonal antibody can easily be determined by one of ordinary skill inthe art so that peptides according to the invention can be preparedusing recombinant DNA technology. There are entities in the UnitedStates which will perform this function commercially, such as ThomasJefferson University and the Scripps Protein and Nucleic Acids CoreSequencing Facility (La Jolla Calif.). For example, the variable regioncDNA can be prepared by polymerase chain reaction from the depositedhybridoma RNA using degenerate or non-degenerate primers (derived fromthe amino acid sequence). The cDNA can be subcloned to producesufficient quantities of double stranded DNA for sequencing byconventional sequencing reactions or equipment.

[0087] Once the nucleic acid sequences of the heavy chain Fd and lightchain variable domains of the deposited MBL monoclonal antibody aredetermined, one of ordinary skill in the art is now enabled to producenucleic acids which encode this antibody or which encode the variousantibody fragments, humanized antibodies, or peptides described above.It is contemplated that such nucleic acids will be operably joined toother nucleic acids forming a recombinant vector for cloning or forexpression of the peptides of the invention. The present inventionincludes any recombinant vector containing the coding sequences, or partthereof, whether for prokaryotic or eukaryotic transformation,transfection or gene therapy. Such vectors may be prepared usingconventional molecular biology techniques, known to those with skill inthe art, and would comprise DNA coding sequences for the CDR3 region andadditional variable sequences contributing to the specificity of theantibodies or parts thereof, as well as other non-specific peptidesequences and a suitable promoter either with (Whittle et al., ProteinEng. 1:499, 1987 and Burton et al., Science 266:1024-1027, 1994) orwithout (Marasco et al., Proc. Natl. Acad. Sci. (USA) 90:7889, 1993 andDuan et al., Proc. Natl. Acad. Sci. (USA) 91:5075-5079,1994) a signalsequence for export or secretion. Such vectors may be transformed ortransfected into prokaryotic (Huse et al., Science 246:1275, 1989, Wardet al., Nature 341: 644-646, 1989; Marks et al., J. Mol. Biol. 222:581,1991 and Barbas et al., Proc. Natl. Acad. Sci. (USA) 88:7978, 991) oreukaryotic (Whittle et al., 1987 and Burton et al., 1994) cells or usedfor gene therapy (Marasco et al., 1993 and Duan et al., 1994) byconventional techniques, known to those with skill in the art.

[0088] As used herein, a “vector” may be any of a number of nucleicacids into which a desired sequence may be inserted by restriction andligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids and phagemids. A cloning vector is one which isable to replicate in a host cell, and which is further characterized byone or more endonuclease restriction sites at which the vector may becut in a determinable fashion and into which a desired DNA sequence maybe ligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. Preferred vectors are those capable ofautonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

[0089] The expression vectors of the present invention includeregulatory sequences operably joined to a nucleotide sequence encodingone of the peptides of the invention. As used herein, the term“regulatory sequences” means nucleotide sequences which are necessaryfor or conducive to the transcription of a nucleotide sequence whichencodes a desired peptide and/or which are necessary for or conducive tothe translation of the resulting transcript into the desired peptide.Regulatory sequences include, but are not limited to, 5′ sequences suchas operators, promoters and ribosome binding sequences, and 3′ sequencessuch as polyadenylation signals. The vectors of the invention mayoptionally include 5′ leader or signal sequences, 5′ or 3′ sequencesencoding fusion products to aid in protein purification, and variousmarkers which aid in the identification or selection of transformants.The choice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art. The subsequentpurification of the peptides may be accomplished by any of a variety ofstandard means known in the art.

[0090] A preferred vector for screening peptides, but not necessarilypreferred for the mass production of the peptides of the invention, is arecombinant DNA molecule containing a nucleotide sequence that codes forand is capable of expressing a fusion peptide containing, in thedirection of amino- to carboxy-terminus, (1) a prokaryotic secretionsignal domain, (2) a peptide of the invention, and, optionally, (3) afusion protein domain. The vector includes DNA regulatory sequences forexpressing the fusion peptide, preferably prokaryotic regulatorysequences. Such vectors can be constructed by those with skill in theart and have been described by Smith et al. (Science 228:1315-1317,1985), Clackson et al. (Nature 352:624-628, 1991); Kang et al. (in“Methods: A Companion to Methods in Enzymology: Vol. 2”, R. A. Lernerand D. R. Burton, ed. Academic Press, NY, pp 111-118,1991); Barbas etal. (Proc. Natl. Acad. Sci. (USA) 88:7978-7982, 1991), Roberts et al.(Proc. Natl. Acad Sci. (USA) 89:2429-2433, 1992)

[0091] A fusion peptide may be useful for purification of the peptidesof the invention. The fusion domain may, for example, include a poly-Histail which allows for purification on Ni+ columns or the maltose bindingprotein of the commercially available vector pMAL (New England BioLabs,Beverly, Mass.). A currently preferred, but by no means necessary,fusion domain is a filamentous phage membrane anchor. This domain isparticularly useful for screening phage display libraries of monoclonalantibodies but may be of less utility for the mass production ofantibodies. The filamentous phage membrane anchor is preferably a domainof the cpIII or cpVIII coat protein capable of associating with thematrix of a filamentous phage particle, thereby incorporating the fusionpeptide onto the phage surface, to enable solid phase binding tospecific antigens or epitopes and thereby allow enrichment and selectionof the specific antibodies or fragments encoded by the phagemid vector.

[0092] The secretion signal is a leader peptide domain of a protein thattargets the protein membrane of the host cell, such as the periplasmicmembrane of gram negative bacteria. A preferred secretion signal for E.coli is a pelB secretion signal. The predicted amino acid residuesequences of the secretion signal domain from two pelB gene producingvariants from Erwinia carotova are described in Lei, et al. (Nature381:543-546, 1988). The leader sequence of the pelB protein haspreviously been used as a secretion signal for fusion proteins (Better,et al., Science 240:1041-1043, 1988; Sastry, et al., Proc. Natl. Acad.Sci (USA) 86:5728-5732, 1989; and Mullinax, et al., Proc. Natl. Acad.Sci. (USA) 87:8095-8099, 1990). Amino acid residue sequences for othersecretion signal peptide domains from E. coli useful in this inventioncan be found in Oliver, In Neidhard, F. C. (ed.), Escherichia coli andSalmonella Typhimurium, American Society for Microbiology, Washington,D.C., 1:56-69 (1987).

[0093] To achieve high levels of gene expression in E. coli, it isnecessary to use not only strong promoters to generate large quantitiesof mRNA, but also ribosome binding sites to ensure that the mRNA isefficiently translated. In E. coli, the ribosome binding site includesan initiation codon (AUG) and a sequence 3-9 nucleotides long located3-11 nucleotides upstream from the initiation codon (Shine, et al.,Nature 254:34, 1975). The sequence, AGGAGGU, which is called theShine-Dalgarno (SD) sequence, is complementary to the 3′ end of E. coli16S rRNA. Binding of the ribosome to mRNA and the sequence at the 3′ endof the mRNA can be affected by several factors:

[0094] (l) The degree of complementarity between the SD sequence and 3′end of the 16S rRNA.

[0095] (ii) The spacing and possibly the DNA sequence lying between theSD sequence and the AUG (Roberts, et al., Proc. Natl. Acad. Sci. (USA)76:760.,1979a: Roberts, et al., Proc. Natl. Acad. Sci. (USA) 76:5596,1979b; Guarente, et al., Science 209:1428, 1980; and Guarente, et al.,Cell 20:543, 1980). Optimization is achieved by measuring the level ofexpression of genes in plasmids in which this spacing is systematicallyaltered. Comparison of different mRNAs shows that there arestatistically preferred sequences from positions −20 to +13 (where the Aof the AUG is position 0) (Gold, et al., Annu. Rev. Microbiol. 35:365,1981). Leader sequences have been shown to influence translationdramatically (Roberts, et al., 1979a, b supra).

[0096] (iii) The nucleotide sequence following the AUG, which affectsribosome binding (Taniguchi, et al., J. Mol. Biol, 118:533, 1978).

[0097] The 3′ regulatory sequences define at least one termination(stop) codon in frame with and operably joined to the heterologousfusion peptide.

[0098] In preferred embodiments with a prokaryotic expression host, thevector utilized includes a prokaryotic origin of replication orreplicon, i.e., a DNA sequence having the ability to direct autonomousreplication and maintenance of the recombinant DNA moleculeextra-chromosomally in a prokaryotic host cell, such as a bacterial hostcell, transformed therewith. Such origins of replication are well knownin the art. Preferred origins of replication are those that areefficient in the host organism. A preferred host cell is E. coli. Foruse of a vector in E. coli, a preferred origin of replication is ColE1found in pBR322 and a variety of other common plasmids. Also preferredis the p15A origin of replication found on pACYC and its derivatives.The ColE1 and p15A replicons have been extensively utilized in molecularbiology, are available on a variety of plasmids and are described bySambrook. et al., Molecular Cloning: A Laboratory Manual, 2nd edition,Cold Spring Harbor Laboratory Press, 1989).

[0099] In addition, those embodiments that include a prokaryoticreplicon preferably also include a gene whose expression confers aselective advantage, such as drug resistance, to a bacterial hosttransformed therewith. Typical bacterial drug resistance genes are thosethat confer resistance to ampicillin, tetracycline, neomycin/kanamycinor chloramphenicol. Vectors typically also contain convenientrestriction sites for insertion of translatable DNA sequences. Exemplaryvectors are the plasmids pUC18 and pUC19 and derived vectors such aspcDNAII available from Invitrogen, (San Diego, Calif.).

[0100] When the peptide of the invention is an antibody including bothheavy chain and light chain sequences, these sequences may be encoded onseparate vectors or, more conveniently, may be expressed by a singlevector. The heavy and light chain may, after translation or aftersecretion, form the heterodimeric structure of natural antibodymolecules. Such a heterodimeric antibody may or may not be stabilized bydisulfide bonds between the heavy and light chains.

[0101] A vector for expression of heterodimeric antibodies, such as theintact antibodies of the invention or the F(ab′)₂, Fab or Fv fragmentantibodies of the invention, is a recombinant DNA molecule adapted forreceiving and expressing translatable first and second DNA sequences.That is, a DNA expression vector for expressing a heterodimeric antibodyprovides a system for independently cloning (inserting) the twotranslatable DNA sequences into two separate cassettes present in thevector, to form two separate cistrons for expressing the first andsecond peptides of a heterodimeric antibody. The DNA expression vectorfor expressing two cistrons is referred to as a dicistronic expressionvector.

[0102] Preferably, the vector comprises a first cassette that includesupstream and downstream DNA regulatory sequences operably joined via asequence of nucleotides adapted for directional ligation to an insertDNA. The upstream translatable sequence preferably encodes the secretionsignal as described above. The cassette includes DNA regulatorysequences for expressing the first antibody peptide that is producedwhen an insert translatable DNA sequence (insert DNA) is directionallyinserted into the cassette via the sequence of nucleotides adapted fordirectional ligation.

[0103] The dicistronic expression vector also contains a second cassettefor expressing the second antibody peptide. The second cassette includesa second translatable DNA sequence that preferably encodes a secretionsignal, as described above, operably joined at its 3′ terminus via asequence of nucleotides adapted for directional ligation to a downstreamDNA sequence of the vector that typically defines at least one stopcodon in the reading frame of the cassette. The second translatable DNAsequence is operably joined at its 5′ terminus to DNA regulatorysequences forming the 5′ elements. The second cassette is capable, uponinsertion of a translatable DNA sequence (insert DNA), of expressing thesecond fusion peptide comprising a secretion signal with a peptide codedby the insert DNA.

[0104] The peptides of the present invention may also, of course, beproduced by eukaryotic cells such as CHO cells, human hybridomas,immortalized B-lymphoblastoid cells, and the like. In this case, avector is constructed in which eukaryotic regulatory sequences areoperably joined to the nucleotide sequences encoding the peptide. Thedesign and selection of an appropriate eukaryotic vector is within theability and discretion of one of ordinary skill in the art. Thesubsequent purification of the peptides may be accomplished by any of avariety of standard means known in the art.

[0105] In another embodiment, the present invention provides host cells,both prokaryotic and eukaryotic, transformed or transfected with, andtherefore including, the vectors of the present invention.

[0106] As used herein, a coding sequence and regulatory sequences aresaid to be “operably joined” when they are covalently linked in such away as to place the expression or transcription of the coding sequenceunder the influence or control of the regulatory sequences. If it isdesired that the coding sequences be translated into a functionalpeptide, two DNA sequences are said to be operably joined if inductionof a promoter in the 5′ regulatory sequences results in thetranscription of the coding sequence and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion to direct the transcription of the coding sequences, or (3)interfere with the ability of the corresponding RNA transcript to betranslated into a protein. Thus, a promoter region would be operablyjoined to a coding sequence if the promoter region were capable ofeffecting transcription of that DNA sequence such that the resultingtranscript might be translated into the desired peptide.

[0107] The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences, as desired.

[0108] According to the methods of the invention, the compositions maybe administered in a pharmaceutically acceptable composition. Ingeneral, pharmaceutically-acceptable carriers for monoclonal antibodies,antibody fragments, and peptides are well-known to those of ordinaryskill in the art. As used herein, a pharmaceutically-acceptable carriermeans a non-toxic material that does not interfere with theeffectiveness of the biological lo activity of the active ingredients,i.e., the ability of the MBL inhibitor to inhibit LCP associatedcomplement activation. Pharmaceutically acceptable carriers includediluents, fillers, salts, buffers, stabilizers, solubilizers and othermaterials which are well-known in the art. Exemplary pharmaceuticallyacceptable carriers for peptides in particular are described in U.S.Pat. No. 5,211,657. The peptides of the invention may be formulated Isinto preparations in solid, semi-solid, liquid or gaseous forms such astablets, capsules, powders, granules, ointments, solutions,depositories, inhalants (e.g., aerosols) and injections, and usual waysfor oral, parenteral or surgical administration. The invention alsoembraces locally administering the compositions of the invention,including as implants.

[0109] According to the methods of the invention the compositions can beadministered by injection by gradual infusion over time or by any othermedically acceptable mode. The administration may, for example, beintravenous, intraperitoneal, intramuscular, intracavity, subcutaneousor transdermal. Preparations for parenteral administration includessterile aqueous or nonaqueous solutions, suspensions and emulsions.Examples of nonaqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil such as olive oil, an injectable organic esterssuch as ethyloliate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, (such as those based on Ringer's dextrose),and the like. Preservatives and other additives may also be present suchas, for example, antimicrobials, antioxidants, chelating agents, andinert gases and the like. Those of skill in the art can readilydetermine the various parameters for preparing these alternativepharmaceutical compositions without resort to undue experimentation.When the compositions of the invention are administered for thetreatment of pulmonary disorders the compositions may be delivered forexample by aerosol.

[0110] The compositions of the invention are administered intherapeutically effective amounts. As used herein, an “effective amount”of the inhibitor of the invention is a dosage which is sufficient toinhibit the increase in, maintain or even reduce the amount ofundesireable LCP associated complement activation. The effective amountis sufficient to produce the desired effect of inhibiting associatedcellular injury until the symptoms associated with the MBL mediateddisorder are ameliorated or decreased. Preferably an effective amount ofthe peptide is an effective amount for preventing cellular injury.Generally, a therapeutically effective amount may vary with thesubject's age, condition, and sex, as well as the extent of the diseasein the subject and can be determined by one of skill in the art. Thedosage may be adjusted by the individual physician or veterinarian inthe event of any complication. A therapeutically effective amounttypically will vary from about 0.01 mg/kg to about 500 mg/kg, weretypically from about 0.1 mg/kg to about 200 mg/kg, and often from about0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily,for one or several days (depending of course of the mode ofadministration and the factors discussed above). A preferredconcentration of the inhibitor is a concentration which is equimolar tothe concentration of MBL in the plasma of a subject. The normal plasmaconcentration of MBL can be assessed clinically. A normal range of MBLis 1-2 μg/ml MBL/plasma.

[0111] One of skill in the art can determine what an effective amount ofan inhibitor is by screening the ability of the inhibitor to inhibit theLCP associated complement activation in an in vitro assay. The activityof the inhibitor can be defined in terms of the ability of the inhibitorto inhibit LCP associated complement activation. An exemplary assay formeasuring the ability of a putative inhibitor of the invention toinhibit LCP associated complement activation is provided in the Examplesand has been discussed above. The exemplary assay is predictive of theability of an inhibitor to inhibit LCP associated complement activationin vivo and, hence, can be used to select inhibitors for therapeuticapplications.

[0112] The MBL inhibitors may be administered in a physiologicallyacceptable carrier. The term “physiologically-acceptable” refers to anon-toxic material that is compatible with the biological systems suchof a tissue or organism. The physiologically acceptable carrier must besterile for in vivo administration. The characteristics of the carrierwill depend on the route of administration. The characteristics of thecarrier will depend on the route of administration.

[0113] The invention further provides detectably labeled, immobilizedand toxin conjugated forms of the peptides, antibodies and fragmentsthereof. The antibodies may be labeled using radiolabels, fluorescentlabels, enzyme labels, free radical labels, avidin-biotin labels, orbacteriophage labels, using techniques known to the art (Chard,Laboratory Techniques in Biology, “An Introduction to Radioimmunoassayand Related Techniques,” North Holland Publishing Company (1978).

[0114] Typical fluorescent labels include fluorescein isothiocyanate,rhodamine, phycoerythrin, phycocyanin, allophycocyanin, andfluorescamine.

[0115] Typical chemiluminescent compounds include luminol, isoluminol,aromatic acridinium esters, imidazoles, and the oxalate esters.

[0116] Typical bioluminescent compounds include luciferin, andluciferase. Typical enzymes include alkaline phosphatase,B-galactosidase, glucose-6-phosphate dehydrogenase, maleatedehydrogenase, glucose oxidase, and peroxidase.

[0117] The invention also includes methods for screening a subject forsusceptibility to treatment with an MBL inhibitor. In one aspect, themethod is accomplished by isolating a mammalian cell from a subject anddetecting the presence of an MBL or an MBL ligand on a surface of themammalian cell. The presence of the MBL indicates that the cell issusceptible to LCP-associated complement activiation, and that thesubject is susceptible to treatment with an MBL inhibitor. The mammaliancell may be isolated by any method known in the art, for instance by abiopsy. Another method for accomplishing the screening assay involvesthe steps of contacting a mammalian cell from the subject with a labeledisolated MBL binding peptide and detecting the presence of an MBL on thesurface of the mammalian cell. This assay may be performed in vitro, exvivo, or in vivo. Many labels which can be used to observe the MBLbinding peptide interacting with the mammalian cell are known in the artunder each of these conditions. For instance, radioactive compounds canbe used in vitro, and other biocompatible labels can be used ex vivo orin vivo. Once the subjects are identified which are susceptible totreatment with an MBL inhibitor, the subjects can then be treatedaccording to the methods of the invention.

[0118] The following examples are provided to illustrate specificinstances of the practice of the present invention and are not to beconstrued as limiting the present invention to these examples. As willbe apparent to one of ordinary skill in the art, the present inventionwill find application in a variety of compositions and methods.

EXAMPLES Example 1 MBL and Complement Deposition on Human CoronaryArteries

[0119] Isolation and Purification of MBL. MBL and associated MASPs werepurified from human plasma. MBL was isolated from human plasma aspreviously described {Tan, Chung, et al. 1996 Biochem. J. 319, 329-332}.Briefly, human plasma was mixed with 7% PEG3500 (w:v). The pellet wascollected by centrifugation and resuspended in TBS-Ca²⁺ [50 mM Tris, 150mM NaCl, 0.05% Tween 20 and 20 mM CaCl₂ at pH 7.8]. The supernatant wasapplied to a mannan-Sepharose column (25 ml, Sigma). The column waswashed with TBS-Ca²⁺ with 109 mM EDTA]. The protein containingsupernatant was calcified to 40 mM calcium and then applied to amaltose-Sepharose column (5 ml). The column was washed with TBS-Ca²⁺ andthen eluted with TBS-Ca²⁺ containing 100 mM N-acetylglucosamine. Westernanalysis and SDS-PAGE established purity for MBL, and the absence of IgGand IgM. Purified MBL and associated MASPs were analyzed by SDS/PAGE.Western blotting was performed to rule out IgG and/or IgM contamination.

[0120] Production of Anti-Human MBL Antibodies. Purified human MBL wasused to immunize rabbits to produce polyclonal anti-human MBL antibodies(Harlow E, et al., Antibodies: A laboratory manual. Cold Spring Harbor,N.Y., Cold Spring Harbor Laboratory, 1988). Adult rabbits were injectedwith 100 μg of MBL emulsified in complete Freund's adjuvant. Boosterimmunizations (100 μg of MBL in incomplete Freund's adjuvant) werestarted 4 wk after the priming immunization and continued at 4 wkintervals. Polyclonal IgG anti-human MBL antibody (R2.2) was purifiedfrom sera-by protein G affinity chromatography.

[0121] Human Coronary Artery Immunohistochemistry. Immunohistochemicalanalysis of MBL, C3d, IgG, IgM, transferrin, and haptoglobin depositionwas performed on tissue specimens from normal (n=14) and atherosclerotichuman epicardial coronary arteries (n=18) obtained at autopsy(Department of Pathology, University of Helsinki, Finland) from patientswho expired secondary to an acute myocardial infarction (MI). Thecontrol specimens were histologically normal coronary arteries obtainedfrom patients who died from non-cardiovascular causes. The mean (±SD) MIage (time difference between the beginning of the clinical episode anddeath) was 5±5 days. The mean (±SD) age of patients suffering from acuteMI was 65±15 years compared to 66±24 years for the control patients.Infarcted myocardium was identified macroscopically at autopsy bydiscolor, pallor, and hyperemia. To improve minacroscopic diagnosis, aslice of non-fixed myocardium was incubated in nitroblue tetrazoliumsolution that leaves the damaged myocardium unstained. Histopathologicalfirst signs of infarction were wavy myocardial fibers and myocytolysisfollowed by signs of coagulation necrosis (i.e., edema, hemorrhage,neutrophil infiltration and pyknosis of nuclei). Infarcts older than 24hr showed signs of total coagulative necrosis with loss of nuclei andstriations together with heavy interstitial neutrophil infiltration.Coronary blood vessel samples for indirect immunofluorescence (IFL)microscopy were snap frozen in liquid nitrogen and stored at −80° C.until analyzed. Frozen sections (4 μm) were air dried and fixed in −20°C. acetone for 10 min. The tissue samples were then incubated for 30 minat 22° C. with either polyclonal rabbit anti-human C3d (Dakopatts,Glostrup, Denmark), MBL (polyclonal R2.2), IgG, IgM, transferrin, orhaptoglobin antibody (all from Behringwerke AG, Germany). After washingwith PBS, the specimens were then stained with an appropriatefluorescein isothiocyanate (FITC)-conjugated secondary antibody.Controls consisted of specimens incubated with nonimmune sera or thesecondary antibody alone. The slides were then mounted with Mowiol andexamined with an Olympus Standard microscope equipped with a filterspecific for FITC-fluorescence.

[0122] Results. Atherosclerotic coronary arteries obtained from patientssuffering from acute MI demonstrated specific MBL and C3d deposits onthe endothelium, intima, and media Immunohistochemical analysis of humancoronary arteries, and in particular, the IFL microscopicaldemonstration of MBL and C3d deposition in an atherosclerotic humancoronary artery was performed. MBL and C3d were observed co-localizedwithin the atherosclerotic lesion. MBL staining of a normal coronaryartery was also performed. Antisera against human transferrin,haptoglobin, IgG, and IgM did not stain normal or atherosclerotic humancoronary arteries.

[0123] Additionally, MBL was observed to co-localize with C3d, withstaining intensity being greatest in ruptured atherosclerotic plaques.Specifically, MBL and C3d deposition appeared to be greatest in thelipid core and surrounding areas of this core in atheroscleroticlesions. No MBL deposits were seen on normal coronary arteries, althoughthe basement membrane sometimes appeared to stain lightly for MBL.Further, antisera against human transferrin, haptoglobin, IgG, and IgMdid not stain normal human coronary arteries or atherosclerotic lesionsin vessels obtained from acute MI patients. Similarly, no staining wasobserved in control experiments in which human coronaries were stainedwith non-immune rabbit serum or with the secondary antibody only. Thesedata demonstrated that MBL co-localized with complement in humancoronary atherosclerotic lesions in patients who have died of acute MI.

Example 2 Endothelial Hypoxia/Reoxygenation Effects MBL Deposition

[0124] Cell Culture. Human umbilical vein endothelial cells (HUVECs)were harvested with 0.1% collagenase (Worthington Biochemical Corp.,Freehold, N.J.) and suspended in Media 199 containing 20%heat-inactivated bovine calf serum (Gibco Life Technologies Inc., GrandIsland, N.Y.). The cells were initially seeded in either 75 cm² flasksor 100 mm Petri dishes (Corning Costar, Cambridge, Mass.), and incubatedat 37° C. in 95% air and 5% CO₂. When confluent, the endothelial cellswere passaged with 0.5% trypsin-EDTA. Endothelial cell purity wasassessed by phase microscopic “cobblestone appearance”, uptake offluorescent acetylated low-density lipoprotein and the presence of vonWillebrand factor. All experiments were conducted on HUVECs duringpassages 1-3.

[0125] MBL-depleted Human Serum (HS). HS was depleted of MBL by affinitychromatography using mannan cross-linked to 4% beaded agarose (SigmaChemical Co., St. Louis, Mo.). All operations were performed at 4° C. HSwas treated with 2 mmol/L ethylenediamine tetraacetate (EDTA) andphenylmethanesulfonyl fluoride (PMSF) to inhibit complement activationand was applied to a mannan column equilibrated with loading buffer(1.25 mmol/L NaCl, 10 mmol/L imidazole, 20 mmol/L CaCl₂, pH 7.8). Theresultant eluent was dialyzed overnight in Hank's buffered salt solutioncontaining Mg²⁺ and Ca²⁺.

[0126] Flow Cytometry. HUVECs were grown to confluence in 100-mm Petridishes coated with gelatin. MBL deposition was measured by flowcytometry in normoxic HUVECs and HUVECs subjected to 24 hr of hypoxiafollowed by 3 hr of reoxygenation in the presence of 30% HS. Afterwashing the cells in Ca²⁺ free or sufficient buffer, the cells werefixed, scraped, and then incubated with 20 μg/ml of monoclonalanti-human MBL antibody (Biodesign, Kennebunk, Me., clone #131-1) orisotype control monoclonal antibody to porcine C5a for 1.5 hr at 4° C.The cells were then washed and incubated with a FITC-conjugated goatanti-mouse IgG secondary antibody for 1 hr at 4° C. MBL deposition onHUVECs was measured by florescence activated cell sorting (FACS) usingthe FACSort flow cytometer (Becton Dickinson, San Jose, Calif.). Allflow cytometry experiments were performed in triplicate.

[0127] Enzyme-Linked Immunoabsorbent Assay (ELISA) Experiments. C3 andMBL specific cell surface ELISAs were developed usingperoxidase-conjugated polyclonal goat anti-human C3 antibody (Cappel,West Chester, Pa.) and monoclonal anti-human MBL antibody (Biodesign,Kennebunk, Me., clone #131-1), respectively. HUVECs were grown toconfluence on 0.1% gelatinized 96-well plastic plates (Corning Costar,Cambridge, Mass.). The plates were then subjected to 0 (normoxia) or 24hr of hypoxia. Hypoxic stress was maintained using a humidified sealedchamber (Coy Laboratory Products, Inc., Grass Lake, Mich.) at 37° C.gassed with 1% O₂, 5% CO₂, balance N₂ (Collard C D, et al.,“Reoxygenation of hypoxic human umbilical vein endothelial cellsactivates the classical complement pathway”, Circulation1997;96:326-333). Following the specified period of normoxia or hypoxia,the cell media were aspirated and 100 μt of one of the following wasadded to each well: 1) 30% HS, 2) Hank's balanced salt solution, 3) 30%HS+3, 30, or 300 mmol/L GluNAc, 4) 30% HS+3, 30, or 300 mmol/LD-inannose, 5) 30% HS+3, 30, 300 mmol/L L-mannose, 6) 30% MBL-depletedHS+3F8 (0, 20, 50 μg/ml)or 7) 30% MBL-depleted HS+0.6 μg/ml MBL.Additionally, 100 μl of purified MBL (3-300 ng/ml) was added to selectwells to form a standard curve for quantitative analysis of MBLdeposition. The cells were then reoxygenated for 3 hr at 37° C. in 95%air and 5% CO₂. The cells were washed and then fixed with 1%paraformaldehyde (Sigma Chemical Co., St. Louis, Mo.) for 30 min. Thecells were then washed and incubated at 4° C. for 1.5 hr with 50 μl ofperoxidase-conjugated polyclonal goat anti-human C3 antibody (1:1000dilution) or monoclonal anti-human MBL antibody (1:1000 dilution). TheMBL ELISA plates were then washed and incubated for 1 hr at 4° C. with50 μl of peroxidase-conjugated polyclonal goat anti-mouse IgG secondaryantibody (1:1000 dilution). After washing the cells, the plates weredeveloped with 50 μl of ABTS(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)), and read(Molecular Devices, Sunnyvale, Calif.) at 405 nm. Background controlsfor the C3 ELISA consisted of cells to which only the anti-human C3antibody was added (i.e., no HS) or cells incubated with 30%heat-inactivated HS. Background controls for the MBL ELISA consisted ofcells to which only secondary antibody and an isotype control monoclonalantibody to porcine C5a were added. Background optical density wassubtracted from all groups. All ELISA experiments were performed 3 timesusing 6 wells per experimental group. Endothelial C3 and MBL depositionon normoxic vs. hypoxic HUVECs was analyzed by two-way analysis ofvariance (ANOVA).

[0128] Results. Flow cytometric analysis (FIG. 2) of endothelial MBLdeposition revealed that the mean fluorescent intensity (MFI) of hypoxicHUVECs (24 hr) reoxygenated (3 hr) in 30% HS was significantly greaterthan normoxic HUVECs or hypoxic HUVECs reoxygenated in buffer alone.Further, MBL deposition was not observed following hypoxia/reoxygenationif the cells were washed in Ca²⁺-free buffer. Thus, MBL deposition onhypoxic/reoxygenated HUVECs was Ca²⁺-dependent.

[0129] In order to further confirm these findings, MBL deposition wasmeasured by ELISA on normoxic HUVECs and HUVECs subjected to 24 hr ofhypoxia followed by 3 hr of reoxygenation in the presence of 30% HS or30% HS treated with 3, 30, or 300 mmol/L of N-acetyl-D-glucosamine(GluNAc) to competitively inhibit MBL deposition. MBL deposition onhypoxic HUVECs reoxygenated in 30% HS was significantly greater(approximately 3-fold increase; p<0.05) than on normoxic HUVECs orHUVECs reoxygenated in HS treated with GluNAc (FIG. 3). Addition ofGluNAc to the HS significantly inhibited MBL deposition onhypoxic/reoxygenated HUVECs in a dose-dependent manner with 3, 30 and300 mmol/L of GluNAc attenuating MBL deposition 40±4%, 71±5% and 96±3%,respectively. Finally, quantitative analysis of the standard curveformed by the addition of purified human MBL (3-300 ng/ml) revealed thatapproximately 3 ng or 8.3×10⁻⁵ fmol of MBL maximally deposits per well(e.g., 48,200±1000 molecules/cell) of hypoxic/reoxygenated HUVECsassuming 2×10⁵ HUVECs/well and a MBL MW of 600 kDa. Thus,hypoxia/reoxygenation increased endothelial MBL deposition.

Example 3 Deposition of iC3b Following Competitive Inhibition of MBL

[0130] HUVEC cell culture and quantitation of iC3b deposition by ELISAwere performed as outlined in Example 2.

[0131] Results. HUVECs were subjected to 0 or 24 hr of hypoxia followedby 3 hr of reoxygenation in the presence of 30% HS or 30% HS treatedwith 3, 30, or 300 mmol/L GluNAc, D-mannose or L-mannose in order toinhibit MBL deposition, LCP activation and iC3b deposition. Depositionof iC3b on hypoxic HUVECs reoxygenated in 30% HS or 30% HS treated withL-mannose was significantly greater (approximately 3-fold;OD₄₀₅=0.14±0.01; p<0.05) than normoxic HUVECs (OD₄₀₅=0.05±0.01) orhypoxic HUVECs reoxygenated in HS treated with GluNAc or D-mannose (FIG.4a). Further, D-mannose, but not L-mannose, inhibited iC3b deposition onhypoxic/reoxygenated HUVECs in dose-dependent manner with 3, 30 and 300mmol/L of D-mannose attenuating iC3b deposition 19±2%, 52±3% and 96±2%,respectively. Thus, these data demonstrated that inhibition of MBLdeposition using GluNAc or D-mannose during reoxygenation significantlyattenuated complement activation and iC3b deposition followingreoxygenation of hypoxic endothelial cells. Further, inhibition of iC3bdeposition with mannose was stereospecific as L-mannose inconcentrations up to 300 mmol/L did not inhibit iC3b deposition (FIG.4a).

Example 4 Deposition of iC3b Following MBL Depletion and Reconstitution

[0132] HUVEC cell culture and quantitation of iC3b deposition by ELISAwere carried out as in Example 2.

[0133] Results. HUVECs were subjected to 0 or 24 hr of hypoxia followedby 3 hr of reoxygenation in the presence of 30% HS, 30% MBL-depleted HSor 30% MBL-depleted HS to which MBL was added back (FIG. 4b). Depositionof iC3b on hypoxic HUVECs reoxygenated in HS was significantly greater(p<0.05) than on normoxic HUVECs. However, iC3b deposition on hypoxicHUVECs reoxygenated in MBL-depleted HS was significantly less (p<0.05)than on hypoxic HUVECs reoxygenated in HS. When MBL was added back tothe MBL-depleted HS, iC3b deposition on HUVECs following 24 hr ofhypoxia and 3 hr of reoxygenation was restored. These data demonstratedthat reoxygenation of hypoxic human endothelial cells activated the LCPleading to increased deposition of iC3b.

Example 5 Complement Hemolytic Assay (CH₅₀) of MBL-Depleted HS

[0134] Methods. Hemolytic assays were completed as previously describedby us {Amsterdam, Stahl, et al., Limitation of reperfusion injury by amonoclonal antibody to C5a during myocardial infarction in pigs, Am. J.Physiol. Heart Circ. Physiol. 1995; 268:H448-H457} {Lennon, Collard, etal., Complement-induced endothelial dysfunction in rabbits: mechanisms,recovery, and gender differences, Am. J. Physiol. Heart Circ. Physiol.,1996; 270:H1924-H1932} {Vakeva, Agah, et al. Myocardial infarction andapoptosis after myocardial ischemia and reperfusion. Role of theterminal complement components and inhibition by anti-C5 therapy.,Circulation 1998; 97:2259-2267}. Briefly, chicken red blood cells weresensitized with sheep anti-chicken antibodies. Serial dilutions of serawere then used to lyse the cells. Hemolytic activity was calculated byusing 0.1% Triton X100 and PBS as positive and negative controls,respectively. Optical density was read at 550 nm on a plate reader.Percent hemolytic activity was calculated as follows:

(Sample OD−PBS)/(Triton OD−PBS)×100=% hemolytic OD

[0135] Samples were run in triplicate and at three determinations pergroup were performed.

[0136] Results. Complement hemolytic assay (CH₅₀) was performed on theMBL-depleted HS in order to demonstrate that MBL depletion did not alterthe classical complement pathway. CH₅₀ of the MBL-depleted HS revealedclassical complement pathway activity similar to that of complete HS(FIG. 5). Similar findings were observed when antibodies 3F8, and 2A9were used. Thus, the decrease in iC3b deposition on hypoxic HUVECsreoxygenated in MBL-depleted serum was not a result of altered classicalpathway complement components (i.e., C1q, C1r, and C1s).

Example 6 Western Blot Analysis of C3 Activation FollowingHypoxia/Reoxygenation Using Purified C2, C3 C4 and MBL

[0137] Western Blot. HUVECs were grown to confluence in 96 well platesand then subjected to normoxia or hypoxia (24 hr). The cells were thenwashed with GVB+ and reoxygenated for 3 hr in the presence of 50 μl ofthe following complement cocktail: MBL (1.2 μg/ml), C2 (8 μg/ml), C3(400 μg/ml), and C4 (200 μg/ml) (C2, C3, and C4 were purchased fromAdvanced Research Technologies; San Diego, Calif.). These complementconcentrations were representative of the concentrations normallypresent in 30% HS. Following reoxygenation, the supernatants werecollected and the protein concentration determined (BioRad, Hercules,Calif.). Five μg of total protein was then resolved by 9% SDS-PAGE underreduced conditions. The gel was then transferred to nitrocellulose,blocked, and probed for the C3 and C3b α′-chain by western blot (CollardC D, “Reoxygenation of hypoxic human umbilical vein endothelial cellsactivates the classical complement pathway”, Circulation1997;96:326-333). Purified C3 and C3b (Advanced Research Technologies;San Diego, Calif.) served as internal standards for MW comparisons ofthe cleaved C3 α′-chain. This experiment was performed 5 times (n=5).

[0138] Results. Western blot analysis of the C3 and C3b α′-chain wasperformed under reduced conditions on the supernatants of normoxic andhypoxic (12 hr) HUVECs reoxygenated (3 hr) in the presence of purifiedC2, C3, C4, and MBL (FIG. 6). A significant increase in the C3b α′-chainband density was observed in the hypoxic/reoxygenated supernatants(Lanes 2 and 4) compared to the normoxic supernatants (Lanes 1 and 3).These results demonstrated LCP-mediated activation of C3 followingendothelial hypoxic/reoxygenation independent of natural antibody or C1.Thus, complement activation following endothelial hypoxia/reoxygenationappeared to be mediated by the LCP and not the classical complementpathway.

Example 7 Microphysiometer Evaluation of HUVEC Receptor-LigandActivation

[0139] Microphysiometry. Changes in HUVEC extracellular acidificationrate (EAR) were evaluated by use of a Cytosensor microphysiometer(Molecular Devices, Sunnyvale, Calif.). HUVECs were grown to 75%confluence on gelatin-coated (1%) transwell capsules and subjected to 24hr of hypoxia followed by 3 hr of reoxygenation. Following 30 min ofequilibration in modified RPMI containing I mmol/L phosphate buffer(Molecular Devices, Sunnyvale, Calif.), the EARs were determined(Gronert K, et al., “Characterization of human neutrophil andendothelial cell ligand-operated extacellular acidification rate bymicrophysiometry: Impact of reoxygenation”, J.Pharmacol.Exp.Ther.1998;285:252-261). HUVECs were perfused with 300-1500 ng/ml of purifiedMBL (dialyzed in the modified RPMI) for 30 sec before the first ratemeasurement and perfusion was maintained for 40 min. As a positivecontrol, the HUVECs were perfused with media alone for 15 min followingMBL exposure and then stimulated with histamine (1 μmol/L, 15 minperfusion) to evoke extracellular acidification. Each concentration ofMBL was analyzed in two independent chambers containing normoxic orhypoxic/reoxygenated HUVECs. The HUVEC response to each MBLconcentration was evaluated in 3 separate experiments (n=3).

[0140] Results. Microphysiometry was performed on normoxic andhypoxic/reoxygenated HUVECs in order to determine if MBL evokedreceptor-mediated changes in the endothelial EAR. Neither perfusion (40min) of normoxic or hypoxic (12 hr)/reoxygenated (3 hr) HUVECs withpurified MBL (300-1500 ng/ml) evoked a change in the EAR, whereas allcells remained responsive to the agonist histamine. Thus, MBL did notevoke receptor-mediated changes in the EAR in normoxic orhypoxic/reoxygenated HUVECs. These data indicated that MBL binding toreoxygenated HUVECs occurred via a MBL ligand and not a classicalreceptor.

Example 8 Preparation and Characterization of Monoclonal Antibodies toHuman MBL

[0141] Female Balb/C mice were initially inoculated (i.p.) with 250 ulof the following mixture: 250 μl Titermax mixed with 100 μg human MBL in250 μl PBS. The following week and for three consecutive weeks the micewere injected with 50 μg hMBL in 250 μl PBS. On the 4th week the micewere injected with 25 μg MBL in 250 μl PBS and the mice were fused 4days later. The fusion protocol was adapted from Current Protocols inImmunology. The splenocytes were fused 1:1 with myelinoma fusion partnerP301 from ATCC using PEG 150 at 50% w/v. The fused cells were plated ata density of 1.25×10⁶/m. with 100 μl/well of a 96 well microtiter plate.The fusion media consisted of Deficient DME high glucose, Pen/Strep(50,000 U pen, 50,000 μg strep per liter), 4 mM L-glutamine, 20% fetalbovine serum, 10% thyroid enriched media, 1% OPI, 1% NEAA, 1% HAT, and50 μM mercaptoethanol. The cells were fed 100 μl/well on day one and100/well media were exchanged on days 2, 3, 4, 7, 9, 11, and 13. Thelast media change before primary screening consisted of HAT substitutedfor the 1% HT. All subsequent feedings were done with fusion media minusthe minus HT or HAT. Screening was done with human MBL plated to plasticELISA plates (96 well plates). Purified hMBL was plated in each well at50 μl volume containing 2 μg/ml MBL in 2% sodium carbonate buffer. Theplates were then blocked with 3% BSA in PBS. Tissue culture media (50μl) was placed in the wells and incubated for 1 hour at roomtemperature. The plates were washed and a secondary HRP labeled goatanti-mouse IgG antibody was used for detection. Colorimetric analysiswas done with ABTS and read at a405 nm. Positive controls consisted of apolyclonal antibody to human MBL. Cells are then grown in-mediaconsisting of the following: DMEM high glucose no-1-glut, sod, pyruvate500 ml (Irvine Scientific #9024), heat inactivated Hyclone 10%, 1%Non-essential amino acids, 4 mM L-gluamine, 100 U/ml penicillin and 100μg/ml streptomycin. All positive wells were then screened for functionin a secondary screen.

[0142] Functional Screen for Anti-MBL Antibodies.

[0143] Methods. The functional screen for inhibition of MBL function byanti-human MBL antibodies was adapted from the literature{Super,Levinsky, et al., The level of mannan-binding protein regulates thebinding of complement-derived opsonins to mannanand zymosan at low serumconcentrations, Clin. Exp. Immunol. 1990; 79:144-150}. Briefly, 100 μlof meannan (0.5 mg/ml in sodium carbonate/bicarbonate buffer, pH 9.6)was added to RIA/EIA plates at 4 C overnight. The plates were thenwashed 3 times in PBS/0.5% Tween pH 7.3, once in PBS and finally inveronal-buffered saline. Human serum is diluted to 4% in VBS containing5 mM Ca²⁺ and Mg²⁺. Diluted sera and tissue culture supernatant orpurified antibody (various concentrations) are then 1:1 to amannan-coated well to yield a final volume or 100 μl at a concentrationof 2% human sera. The plate is then incubated at 37 C for 30 min.Positive and negative controls consist of human sera without and with100 mM N-acetlglucosamine (GluNac). The plates are then washed fourtimes in PBS/Tween. The plates are then incubated with an anti-human C3polyclonal antibody coupled with HRP (1 hours at RT), washed anddeveloped with ABTS and read at 405 nm.

[0144] Results. Antibody production and characterization. Following aprimary screen using a solid phase antibody-capture ELISA, we identified11 clones that recognized human MBL. After limiting dilution andisotyping, we identified eight mAbs that recognized human MBL in anantibody-capture ELISA. Clones 3F8, 2A9, and hMBL1.2 were isotyped asmouse IgG_(1k), where as clone 1C10 was a mouse IgG_(2b). The otherhybridomas produced IgM antibodies and were not included in this study.

[0145] Western blot analysis was used to determine that the mAbsrecognized MBL. As shown in FIG. 7, antibodies 2A9 (Lane 1), hMBL1.2(Lane 2), 1C10 (Lane 3) or 3F8 (Lane 4) recognized purified and reducedhuman MBL [i.e., molecular weight (MW) ˜32 kD]. Thus, these antibodiesare specific for human MBL. Clones hMBL1.2, 2A9 and 3F8 have beendeposited at the International Despository Authority with ATCCdesignations of HB-12619, HB-12620 and HB-12621, respectively.

[0146] The most potent inhibitor of MBL induced complement activiation,N-acetylglucosamine (GluNAc) inhibited C3 deposition to plastic inmannan coated plates in a dose-dependent manner with an EC50 ofapproximately 1 nM. Similarly, 2A9 and hMBL 1.2 inhibited C3 depositionwith and EC50 of approximately 30 and 50 nM, respectively. An isotypecontrol antibody that recognizes MBL by solid phase ELISA did notinhibit MBL dependent C3 deposition. Thus, these antibodies areapproximately 10⁵-10⁶ times more potent than GluNAc. The data represent3 separate experiments with at least 4 observations per experiment.HUBECs were hypoxic for 24 hours and then reoxygenated in 30% humansera. iC3b deposition was then normalized to normoxic cells. Anapproxinate 190% increase in iC3b deposition on hypoxic cells wasobserved following reoxygenation (FIG. 8). 3F8 attenuated iC3bdeposition on hypoxic/reoxygenated HUVECs in a dose-dependent manner.These data demonstrate that specific inhibition of MBL with an antibodyattenuates complement activation and iC3b deposition followinghypoxia/reoxygenation of human endothelial calls. *p<0.05 compared toall groups; n+2.

Example 9

[0147] Complement activation and deposition following HUVEC oxidativestress. To characterize further the functional properties of these novelmAbs and to demonstrate specifically the role of MBL in complementactivation following oxidative stress of human endothelial cells, weassessed MBL and C3 deposition on hypoxic human endothelial cellsfollowing reoxygenation in human sera.

[0148] Western blot analysis. To demonstrate the complement inhibitoryaction of these anti-human MBL mAbs, hypoxic HUVECs were reoxygenated inhuman sera treated with PBS (vehicle), 3F8, hMBL1.2, 2A9, or 1C10 (50μg/ml final concentration). Cell membrane bound proteins were resolvedby SDS-PAGE under reduced conditions, transferred to membranes, andanalyzed for human C3dg (i.e., part of the α-chain of iC3b). The α- andβ-chain of iC3b were the only C3 stainable bands present on the cellularmembranes. A representative C3dg band for vehicle, 3F8-, hMBL1.2-, 2A9-and 1C10-treated cells was observed. We observed a significant decreasein C3dg band intensity on cells reoxygenated in human sera treated witheither 3F8, 2A9 or hMBL1.2. However, the non-functional clone, 1C10, didnot decrease iC3b deposition (i.e., C3dg band intensity) on theendothelial membranes. These data further support the role ofMBL-dependent complement activation following reoxygenation of hypoxicHUVECs. Further, these data confirm that clone 1C10 is an isotypecontrol mAb that does not functionally inhibit MBL.

[0149] Confocal microscopy studies. Dual labeling for MBL and C3deposition on normoxic and hypoxic HUVECs was performed to demonstrateco-localization of these complement components and MBL-dependentcomplement pathway activation. Normoxic and hypoxic HUVECs werereoxygenated in 30% HS treated with and without mAb 3F8 (5 μg/ml) or1C10 (50 μg/ml). MBL (blue), C3 (green) and nuclei (red) were thenstained on the same slide and anzlyzed by immunofluorescent confocalmicroscopy. Small amounts of C3 and MBL staining were observed undernormoxic conditions, confirming our finding of low level C3 depositionunder normoxic conditions, confirming our finding of low level C3deposition under normoxic conditions. C3 and MBL staining onhypoxic/reoxygenated HUVECs was significantly greater than normoxicHUVECs. Clone 1C10 failed to inhibit C3 or MBL deposition followingoxidative stress. C3 and MBL staining was significantly decreased onhypoxic/reoxygenated HUVECs treated with mAb 3F8 (5 μg/ml) to levelsbelow those observed under normoxic conditions (similar results wereobserved with mAbs hMBL1.2 or 2A9). It was observed that MBL and C3co-localize on human endothelial cells under the conditions outlinedabove. These data demonstrate that functional inhibition of MBL with amAb attenuates C3 deposition following oxidative stress of humanendothelial cells.

Example 10

[0150] Methods: VCAM-1 ELISA. Briefly, HUVECs were grown to confluenceon 0.1% gelatinized 96-well plastic plates and then subjected to 0 or 12hr of hypoxia. The cell media was then aspirated and HBSS, 30% HS or 30%HS treated with 3F8 (5 μg/ml) was added to each well. The cells werethen reoxygenated for 3 hr at 37° C. in 95% air and 5% CO2. The cellswere washed, fixed, washed again, and incubated at 4° C. for 1.5 hr withthe anti-human VCAM-1 mAb (clone 6G10 obtained from the DevelopmentalStudies Hybridoma Bank, University of Iowa, Iowa City, Iowa). Aperoxidase-conjugated goat anti-mouse secondary antibody (Cappel, WestChester, Pa.) was then used. An inappropriate isotype control antibody(mAb GSI to porcine C5a) was used to assess background optical densityand fluorescence was subtracted from the data. These experiments (6wells per experimental group) were performed 3 times (n=3).

[0151] Results: Inhibition of VCAM-1 expression following oxidativestress. We have demonstrated that oxidative stress of HUVECs activatescomplement and results in C5b-9 dependent VCAM-1 induction. Thus, weexamined VCAM-1 expression by ELISA to demonstrate further thefunctional significance of MBL inhibition. As shown in FIG. 9 andconfirming our own findings, reoxygenation of hypoxic HUVECs in 30% HStreated with PBS (vehicle) resulted in a significant increase in VCAM-1protein expression. Treatment of 30% HS with 3F8 (5 μg/ml) significantlyattenuated VCAM-1 expression. Since VCAM-1 expression in this model ismediated by C5b-9, these data demonstrated that C5b-9 formation isdependent on MBL deposition and lectin pathway activation.

[0152] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by examplesprovided, since the examples are intended as a single illustration ofone aspect of the invention and other functionally equivalentembodiments are within the scope of the invention. Various modificationsof the invention in addition to those shown and described herein willbecome apparent to those skilled in the art from the foregoingdescription and fall within the scope of the appended claims. Theadvantages and objects of the invention are not necessarily encompassedby each embodiment of the invention.

[0153] All references, patents and patent publications that are recitedin this application are incorporated in their entirety herein byreference.

[0154] Deposits: Hybridoma 3F8, 2A9, and hMBL1.2 were deposited on Dec.4, 1998 with the American Type Culture Collection (ATCC) as ATCCAccession Nos. HB-12621, HB-12620, and HB-12619, respectively, under theterms of the Budapest Treaty.

[0155] The ATCC is a depository affording permanence of the deposit andready accessibility thereto by the public if a patent is granted. Allrestrictions on the availability to the public of the material sodeposited will be irrevocably removed upon the granting of a patent. Thematerial will be available during the pendency of the patent applicationto one determined by the Commissioner to be entitled thereto under 37C.F.R §1.14 and 35 U.S.C §122. The deposited material will be maintainedwith all the care necessary to keep it viable and uncontaminated for aperiod of at least five (5) years after the most recent request for thefurnishing of a sample of the deposited hybridomas, and in any case, fora period of at least thirty (30) years after the date of deposit or forthe enforceable life of the patent, whichever period is longer.Applicants acknowledge its duty to replace the deposit should thedepository be unable to furnish a sample when requested due to thecondition of the deposit.

We claim:
 1. A method for inhibiting lectin complement pathway (LCP)associated complement activation, comprising administering an effectiveamount of a mannose binding lectin (MBL) inhibitor to inhibit LCPassociated complement activation in a subject.
 2. The method of claim 1,wherein the LCP associated complement activation mediates a cellularinjury.
 3. The method of claim 2, wherein the cellular injury mediatedby LCP associated complement activation contributes to tissue injuryassociated with atherosclerosis.
 4. The method of claim 2, wherein thecellular injury mediated by LCP associated complement activationcontributes to-tissue injury associated with the pulmonary system. 5.The method of claim 4, wherein the MBL inhibitor is administered to thesubject by an aerosol route of delivery.
 6. The method of claim 2,wherein the cellular injury mediated by LCP associated complementactivation contributes to tissue injury associated with arthritis,myocardial infarction, ischemia, reperfusion, transplantation,cardiopulmonary bypass (CPB), stroke, acute respiratory distresssyndrome (ARDS), systemic lupus erythematosus (SLE), lupus, or dialysis.7. The method of claim 2, wherein the cellular injury mediated by LCPassociated complement activation contributes to tissue injury associatedwith ischemia.
 8. The method of claim 2, further comprisingadministering to the subject a therapeutic treatment for treating an MBLmediated disorder associated with the cellular injury mediated by LCPassociated complement activation.
 9. The method of claim 8, wherein thetherapeutic treatment is a drug.
 10. The method of claim 8, wherein thetherapeutic treatment comprises revascularizing a coronary artery. 11.The method of claim 10, wherein the revascularizing of a coronary arteryis achieved by a method comprising percutaneous transluminal coronaryangioplasty.
 12. The method of claim 1, wherein a mammalian cell with asurface exposed MBL ligand is contacted with the MBL inhibitor.
 13. Themethod of claim 1, wherein the MBL inhibitor binds to MBL.
 14. Themethod of claim 13, wherein the MBL inhibitor is an MBL binding peptide,protein, antibody, or antibody fragment.
 15. The method of claim 13,wherein the MBL inhibitor binds to a human MBL epitope.
 16. The methodof claim 15, wherein the human MBL epitope is a region of MBL whichinteracts with a monoclonal antibody produced by: i) hybridoma_((3F8))deposited under ATCC Accession No. HB-12621, ii) hybridoma_((2A9))deposited under ATCC Accession No. HB-12620, or iii)hybridoma_((hMBL1.2)) deposited under ATCC Accession No. HB-12619. 17.The method of claim 13, wherein the MBL inhibitor competes for bindingto MBL with a monoclonal antibody produced by: i) hybridoma_((3F8))deposited under ATCC Accession No. HB-12621, ii) hybridoma_((2A9))deposited under ATCC Accession No. HB-12620, or iii)hybridoma_((hMBL1.2)) deposited under ATCC Accession No. HB-12619. 18.The method of claim 13, wherein the MBL inhibitor comprises an MBLbinding CDR3 region or functional variant thereof.
 19. The method ofclaim 18, wherein the CDR3 region is of a monoclonal antibody producedby: i) hybridoma_((3F8)) deposited under ATCC Accession No. HB-12621,ii) hybridoma_((2A9)) deposited under ATCC Accession No. HB-12620, oriii) hybridoma_((hMBL1.2)) deposited under ATCC Accession No. HB-12619.20. The method of claim 18, wherein the MBL inhibitor further comprisesa CDR2 region or a functional variant thereof.
 21. The method of claim18, wherein the MBL inhibitor further comprises a CDR1 region or afunctional variant thereof.
 22. The method of claim 13, wherein the MBLinhibitor is an antibody fragment.
 23. The method of claim 22, whereinthe antibody fragment is an antibody fragment selected from the groupconsisting of an F(ab′)₂ fragment, an Fd fragment, an Fv fragment, andan Fab fragment.
 24. The method of claim 22, wherein the antibodyfragment is of a monoclonal antibody produced by: i) hybridoma_((3F8))deposited under ATCC Accession No. HB-12621, ii) hybridoma_((2A9))deposited under ATCC Accession No. HB-12620, or iii)hybridoma_((hMBL1.2)) deposited under ATCC Accession No. HB-12619. 25.The method of claim 13, wherein the MBL inhibitor is an antibody. 26.The method of claim 25, wherein the antibody is a monoclonal antibody.27. The method of claim 26, wherein the monoclonal antibody is producedby: i) hybridoma_((3F8)) deposited under ATCC Accession No. HB-12621,ii) hybridoma_((2A9)) deposited under ATCC Accession No. HB-12620, oriii) hybridoma_((hMBL1.2)) deposited under ATCC Accession No. HB-12619.28. The method of claim 25, wherein the antibody is a single-chainantibody.
 29. The method of claim 25, wherein the antibody is ahumanized antibody.
 30. The method of claim 1, wherein the MBL inhibitorinhibits C3b deposition.
 31. The method of claim 30, wherein the MBLinhibitor is an MBL binding peptide, protein, antibody, or antibodyfragment and inhibits C3b deposition with an EC50 of between 10⁻⁹ to10⁻⁷ mol/L.
 32. The method of claim 1, wherein the method furtherinhibits VCAM-1 expression.
 33. The method of claim 1, wherein the MBLinhibitor binds to a mannose-binding lectin-associated serine protease(MASP) or mannan.
 34. The method of claim 33, wherein the MBL inhibitoris a peptide, protein, antibody, or antibody fragment.
 35. The method ofclaim 34, wherein the antibody or antibody fragment is a monoclonalantibody or monoclonal antibody fragment.
 36. The method of claim 34,wherein the antibody or antibody fragment is humanized.
 37. The methodof claim 34, wherein the antibody or antibody fragment is a single-chainantibody.
 38. The method of claim 33, wherein the MASP is MASP-1 orMASP-2.
 39. The method of claim 1, wherein the method is a screeningassay.
 40. A method for inhibiting cellular injury in a subject,comprising administering an effective amount of an MBL inhibitor toinhibit cellular injury in the subject.
 41. The method of claim 40,wherein the cellular injury contributes to tissue injury associated withatherosclerosis.
 42. The method of claim 40, wherein the cellular injurycontributes to tissue injury associated with the pulmonary system. 43.The method of claim 42, wherein the MBL inhibitor is administered to thesubject by an aerosol route of delivery.
 44. The method of claim 40,wherein the cellular injury contributes to tissue injury associated witharthritis, myocardial infarction, ischemia, reperfusion,transplantation, CPB, stroke, ARDS, SLE, lupus, or dialysis.
 45. Themethod of claim 40, wherein the cellular injury contributes to tissueinjury associated with ischemia.
 46. The method of claim 40, furthercomprising administering to the subject a therapeutic treatment fortreating an MBL mediated disorder associated with the cellular injury.47. The method of claim 46, wherein the therapeutic treatment is a drug.48. The method of claim 46, wherein the therapeutic treatment comprisesrevascularizing a coronary artery.
 49. The method of claim 48, whereinthe revascularizing of a coronary artery is achieved by a methodcomprising percutaneous transluminal coronary angioplasty.
 50. Themethod of claim 40, wherein the MBL inhibitor inhibits MBL deposition ona mammalian cell with a surface exposed MBL ligand.
 51. The method ofclaim 40 or 50, wherein the MBL inhibitor binds MBL, MASP or mannan. 52.The method of claim 51, wherein the MBL inhibitor is a peptide, protein,antibody, or antibody fragment.
 53. The method of claim 52, wherein theantibody or antibody fragment is a monoclonal antibody or a monoclonalantibody fragment.
 54. The method of claim 52, wherein the antibody orantibody fragment is humanized.
 55. The method of claim 52, wherein theantibody or antibody fragment is a single-chain antibody.
 56. The methodof claim 51, wherein the MASP is MASP-1 or MASP-2.
 57. The method ofclaim 51, wherein the MBL inhibitor binds to a human MBL epitope. 58.The method of claim 57, wherein the human MBL epitope is a region of MBLwhich interacts with a monoclonal antibody produced by: i)hybridoma_((3F8)) deposited under ATCC Accession No. HB-12621, ii)hybridoma_((2A9)) deposited under ATCC Accession No. HB-12620, or iii)hybridoma_((hMBL1.2)) deposited under ATCC Accession No. HB-12619. 59.The method of claim 52, wherein the MBL binding peptide, protein,antibody, or antibody fragment competes for binding to MBL with amonoclonal antibody produced by: i) hybridoma_((3F8)) deposited underATCC Accession No. HB-12621, ii) hybridoma_((2A9)) deposited under ATCCAccession No. HB-12620, or iii) hybridoma_((hMBL1.2)) deposited underATCC Accession No. HB-12619.
 60. The method of claim 40, wherein the MBLinhibitor inhibits C3b deposition.
 61. The method of claim 60, whereinthe MBL inhibitor is an MBL binding peptide, protein, antibody, orantibody fragment and inhibits C3b deposition with an EC50 of between10⁻⁹ to 10⁻⁷ mol/L.
 62. The method of claim 40, wherein the methodfurther inhibits VCAM-1 expression.
 63. The method of claim 51, whereinthe MBL inhibitor has an MBL binding CDR3 region or functional variantthereof.
 64. The method of claim 63, wherein the CDR3 region is of amonoclonal antibody produced by: i) hybridoma_((3F8)) deposited underATCC Accession No. HB-12621, ii) hybridoma_((2A9)) deposited under ATCCAccession No. HB-12620, or iii) hybridoma_((hMBL1.2)) deposited underATCC Accession No. HB-12619.
 65. The method of claim 63, wherein the MBLinhibitor further comprises a CDR2 region or a functional variantthereof.
 66. The method of claim 63, wherein the MBL inhibitor furthercomprises a CDR1 region or a functional variant thereof.
 67. The methodof claim 40, wherein the MBL inhibitor is an antibody fragment.
 68. Themethod of claim 67, wherein the antibody fragment is an antibodyfragment selected from the group consisting of an F(ab′)₂ fragment, anFd fragment, an Fv fragment, and an Fab fragment.
 69. The method ofclaim 68, wherein the antibody fragment is of a monoclonal antibodyproduced by: i) hybridoma_((3F8)) deposited under ATCC Accession No.HB-12621, ii) hybridoma_((2A9)) deposited under ATCC Accession No.HB-12620, or iii) hybridoma_((hMBL1.2)) deposited under ATCC AccessionNo. HB-12619.
 70. The method of claim 40, wherein the MBL inhibitor isan antibody.
 71. The method of claim 70, wherein the antibody is amonoclonal antibody.
 72. The method of claim 71, wherein the monoclonalantibody is produced by: i) hybridoma_((3F8)) deposited under ATCCAccession No. HB-12621, ii) hybridoma_((2A9)) deposited under ATCCAccession No. HB-12620, or iii) hybridoma_((hMBL1.2)) deposited underATCC Accession No. HB-12619.
 73. The method of claim 70, wherein theantibody is a single-chain antibody.
 74. The method of claim 70, whereinthe antibody is a humanized antibody.
 75. The method of claim 40,wherein the method is a screening assay.